JP2000270892A - Detection of bacillus - Google Patents
Detection of bacillusInfo
- Publication number
- JP2000270892A JP2000270892A JP11083590A JP8359099A JP2000270892A JP 2000270892 A JP2000270892 A JP 2000270892A JP 11083590 A JP11083590 A JP 11083590A JP 8359099 A JP8359099 A JP 8359099A JP 2000270892 A JP2000270892 A JP 2000270892A
- Authority
- JP
- Japan
- Prior art keywords
- bacteria
- phage
- solution
- labeled
- bacterium
- 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
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、例えば大腸菌など
の細菌を検出する方法に関するものである。[0001] The present invention relates to a method for detecting bacteria such as E. coli.
【0002】[0002]
【従来の技術】細菌をはじめとする微生物はあらゆる環
境に存在し、人間の生活と密接な関係を保っている。し
かし微生物の中には人間あるいは家畜に疾病をもたらす
ものもあり、病原性微生物として人間あるいは家畜への
感染に注意が払われている。病原性微生物の中でも特に
細菌は増殖が速く、低温、高温、乾燥などの環境条件に
影響を受けにくく、そのため人間の周りに持続的に存在
し、一度好条件を得れば爆発的に数を増やして感染症の
原因となる。これらのことから、食品や飲料水(水道水
を含む)のように人間の体内に直接摂取されるものにつ
いてはもちろんのこと、食品の製造工程で利用される
水、食器等の洗浄に利用される水、河川、湖沼、海、プ
ール、浴場、その他の修景親水利用のためのアメニティ
用水など、人が身体を接触させる水、下水放流水など、
人と接触する機会がある水については、細菌に対する適
正な管理が望まれる。2. Description of the Related Art Bacteria and other microorganisms exist in various environments and maintain a close relationship with human life. However, some microorganisms cause disease in humans or livestock, and attention has been paid to human or livestock infection as a pathogenic microorganism. Among pathogenic microorganisms, bacteria are particularly fast-growing, less susceptible to environmental conditions such as low temperature, high temperature, and dryness. Increase and cause infection. From these facts, it is used not only for foods and drinking water (including tap water) that are directly ingested into the human body, but also for washing water and dishes used in food manufacturing processes. Such as water, rivers, lakes, marshes, seas, pools, baths, and other amenities for the use of scenic water, such as water for human contact and sewage discharge water.
For water that has a chance to come into contact with humans, proper management of bacteria is desired.
【0003】以上のことを前提として、食品、飲料とな
る水道水については「大腸菌群は検出されないこと」と
いう基準、プール水では「10mlの水を5本培養し大
腸菌群陽性の管が2本以下であること」という基準が定
められ、また、修景親水利用の水は「1ml中10個以
下」、一般排水は「1ml中3000個以下」が望まし
いという目標値が設けられている。このように、腸内細
菌特に大腸菌および大腸菌群の有無を調べる項目につい
ての管理基準が、上記各種の利用水(食品、飲料水、あ
るいは親水施設のアメニティ用水等)毎に定められてい
るのは、細菌による感染症が、人あるいは動物を起源と
する細菌によって引き起こされる疾病の頻度が最も高
く、被害も拡大しやすいことから考えれば妥当なところ
である。On the premise of the above, tap water used as food and drink is based on the criterion that "Escherichia coli group should not be detected". The following values are set. The target values are set such that the water for use in the scenic landscape is preferably "10 or less per 1 ml", and the general wastewater is preferably "3000 or less per 1 ml". As described above, the management criteria for items to check for the presence of enterobacteria, especially Escherichia coli and Escherichia coli, are set for each of the above-mentioned various types of water used (food, drinking water, or water for amenities at hydrophilic facilities, etc.). This is reasonable given that bacterial infections are most frequently caused by bacteria of human or animal origin, and the damage is likely to spread.
【0004】ところで、例えば上述のような食品、飲料
水(水道水)、プール水、修景親水用水などを管理する
のに必要な細菌の検出は、従来一般的には培養法により
実施されている。この培養法は、例えば大腸菌や大腸菌
群を例として言えば、これを他の一般細菌と区別して検
出する工夫が必要であるため、例えば、大腸菌や大腸菌
群が選択的に増殖する培地、あるいは増殖すると色調が
変化したりコロニーの色に特殊な色が生じる工夫がされ
た培地を用いて実施される。しかしこの方法は培養によ
って菌が増殖するまでに時間がかかり、加えて、増殖し
た細菌が大腸菌あるいは大腸菌群であるかどうかの判定
法が煩雑であるため最終的な判定までに数日以上の時間
がかかるという問題がある。また一般的な最大希釈倍率
から求める方法による場合は、おおよその定量推定値が
求められるにすぎず、定量性の精度は乏しいという問題
がある。[0004] By the way, for example, the detection of bacteria necessary for controlling food, drinking water (tap water), pool water, water for landscaping and the like as described above has conventionally been generally carried out by a culture method. I have. In this culture method, for example, in the case of Escherichia coli or Escherichia coli group, for example, it is necessary to devise a method of detecting this in distinction from other general bacteria. Then, the culture is performed using a medium whose color tone is changed or a special color is generated in the color of the colony. However, this method requires a long time for the bacteria to grow by culturing, and in addition, the method for determining whether the grown bacteria are Escherichia coli or Escherichia coli is complicated, so that it takes more than several days until the final determination. There is a problem that it takes. In addition, in the case of using a method of obtaining from a general maximum dilution factor, there is a problem that only an approximate quantitative estimation value is obtained, and the accuracy of quantitative performance is poor.
【0005】このような単純な培養法とは別に、簡略化
した検出方法として特異酵素基質培地法も知られてい
る。この方法は、例えば大腸菌や大腸菌群に特異的に含
まれる酵素に対する基質の存在下で試料を培養し、陽性
であれば特徴的な色や蛍光が生じるように工夫した方法
である。[0005] Apart from such a simple culture method, a specific enzyme substrate medium method is also known as a simplified detection method. In this method, for example, a sample is cultured in the presence of a substrate for an enzyme specifically contained in Escherichia coli or an Escherichia coli group, and if positive, a characteristic color or fluorescence is generated.
【0006】上記方法の他にも、PCR法(ポリメラー
ゼ・チェーン・リアクション法)、ATPを測る方法、
更に、特開平8−154700号公報で提案の放射性同
位元素,酵素を標識としてバクテリオファージ表面に結
合する方法、レポータ遺伝子をバクテリオファージのD
NAに遺伝子組換えで組み込む形で標識する方法も知ら
れている。In addition to the above methods, a PCR method (polymerase chain reaction method), a method for measuring ATP,
Furthermore, a method of binding a bacteriophage to the surface of a bacteriophage using a radioisotope and an enzyme as a label proposed in JP-A-8-154700,
A method of labeling NA by genetic recombination is also known.
【0007】しかし、上述した従来法はいずれも、実際
の実施場面から考えると、処理の迅速性、処理操作の簡
便性、検出精度の高さなどの種々の点から極めて解決困
難な問題が以下のようにある。However, in the conventional methods described above, problems that are extremely difficult to solve from various points such as quickness of processing, simplicity of processing operation, high detection accuracy, and the like are considered as follows from the viewpoint of actual implementation. There is like.
【0008】例えば、特異酵素基質培地法は、単純な培
養法に比べて、大腸菌および大腸菌群の定性試験が24
時間程で実施できる利点があるものの、大腸菌や大腸菌
群についても全てを検出することはできないし、定量試
験を行うとすれば最大希釈倍率で陽性を示す値から求め
る形式で行うしかなく、測定法に由来する定量検出の精
度に限界がある。For example, the qualitative test for Escherichia coli and Escherichia coli is more difficult in the specific enzyme substrate medium method than in the simple culture method.
Although there is an advantage that it can be performed in about a time, it is not possible to detect all Escherichia coli and coliforms, and if a quantitative test is to be performed, it must be performed in the form of obtaining the positive value at the maximum dilution factor, and the measurement method There is a limit to the accuracy of quantitative detection derived from
【0009】PCR法は、感度は鋭敏であるが、死滅し
ていて実際には無害な細菌も検出してしまい、死菌,生
菌の区別ができないという問題がある他、定量性も期待
できない。Although the PCR method has a high sensitivity, it detects dead and actually harmless bacteria, so that there is a problem that dead bacteria and live bacteria cannot be distinguished from each other, and quantitativeness cannot be expected. .
【0010】ATP法は、操作が簡単でしかも生菌だけ
を特異的に検出できる点で優れているが、検出感度が低
い(1000個以上の細菌でないと検出できない)とい
う問題があり、これに加えて、細菌を特定して検出でき
ないという、用途によっては致命的な問題がある。[0010] The ATP method is excellent in that it is easy to operate and can specifically detect only live bacteria, but has a problem that its detection sensitivity is low (it cannot be detected unless there are more than 1,000 bacteria). In addition, there is a fatal problem in some applications that bacteria cannot be identified and detected.
【0011】また、前記特開平8−154700号公報
に記載の方法のうち、ファージ表面に放射性同位元素
(アイソトープ)又は酵素を標識して用いる標識法は、
処理の操作性、検出精度に問題がある。すなわち、放射
性同位元素を用いる方法では、細菌に吸着したファージ
がもつ放射性同位元素標識由来の信号と、非吸着のファ
ージがもつ同標識由来の信号を区別することができない
から、これら吸着,非吸着のファージを物理的に分離す
る余分な操作が必要となる。またこれに加えて、細菌
(あるいはろ過膜等の分離手段)に非特異的に吸着した
ファージに標識した放射性同位元素をカウントしてしま
うという致命的な誤検出の虞がある。また、酵素を標識
として用いる方法は、放射性同位元素を用いる場合と同
じように非特異的な吸着がある問題に加えて、微小量の
酵素の活性を利用する方法であるので、酵素活性の作用
を受ける基質に現れる現象から微小量の酵素量を精度よ
く検出することが必ずしも容易でなく、定量検出精度を
向上させるための新たな技術開発が必要になるという問
題がある。[0011] Among the methods described in the above-mentioned JP-A-8-154700, a labeling method using a radioisotope (isotope) or an enzyme on the phage surface by labeling the phage surface is as follows:
There is a problem in processing operability and detection accuracy. That is, in the method using a radioisotope, the signal derived from the radioisotope label of the phage adsorbed on the bacteria cannot be distinguished from the signal derived from the label of the non-adsorbed phage. Extra operation to physically separate the phage is required. In addition, there is a risk of fatal erroneous detection in which radioactive isotopes labeled on phages non-specifically adsorbed to bacteria (or separation means such as a filtration membrane) are counted. In addition, the method of using an enzyme as a label has the problem of non-specific adsorption as in the case of using a radioisotope, and also uses a small amount of enzyme activity. There is a problem that it is not always easy to accurately detect a minute amount of an enzyme due to a phenomenon appearing on a substrate to be subjected to the reaction, and that a new technology development for improving the quantitative detection accuracy is required.
【0012】更に、レポーター遺伝子を用いる方法は、
細菌毎に特異的なファージ夫々について遺伝子組換え体
のレポーター遺伝子を調製する必要があり、このレポー
タ遺伝子を含むファージ核酸の複製,転写,翻訳を行わ
せ、産生蛋白質を検出する操作が必要となるため、汎用
性は殆ど期待できない。Further, a method using a reporter gene is as follows:
It is necessary to prepare a transgenic reporter gene for each phage specific to each bacterium, and it is necessary to perform replication, transcription, and translation of the phage nucleic acid containing this reporter gene, and to perform an operation to detect the produced protein. Therefore, versatility can hardly be expected.
【0013】以上のように、従来知られている種々の方
法はいずれも問題があり、特に、迅速性、操作の簡便
性、検出精度、検出感度などの点でいまだ解決すべき課
題が多い。As described above, all of the conventionally known methods have problems, and there are still many problems to be solved particularly in terms of speed, simplicity of operation, detection accuracy, detection sensitivity, and the like.
【0014】そこで本発明者は、上記のような従来法の
問題点を解消し、細菌そのものを特異的に標識付けする
ことで、特定細菌の検出・判定を可能としながら、測定
の簡易化、短時間化、効率化、高感度化を図ることがで
きる方法として、バクテリオファージの核酸(DNA)
(以下「ファージDNA」という)に色素又は蛍光物質
を標識したバクテリオファージ(以下「ファージ」とい
う)を用いる方法を既に提案した(特願平9−2409
19号)。Therefore, the present inventor has solved the above-mentioned problems of the conventional method, and has made it possible to detect and judge a specific bacterium by specifically labeling the bacterium itself. Bacteriophage nucleic acid (DNA) is a method that can shorten the time, increase the efficiency, and increase the sensitivity.
A method using a bacteriophage (hereinafter, referred to as “phage”) labeled with a dye or a fluorescent substance (hereinafter referred to as “phage DNA”) has already been proposed (Japanese Patent Application No. 9-2409).
No. 19).
【0015】この提案に係わる発明は次の特徴がある。
すなわち、細菌ウイルスであるファージは宿主細菌に対
して特異的に結合し、菌の膜電位に依存して生細胞に対
してのみその細胞内にファージDNAを注入し、また注
入されたファージDNAは、空間的に広い細胞内で拡散
(展開)するという性質を有しているために、ファージ
DNAを色素、蛍光物質で標識しておけば、宿主細菌全
体が染色されて大きな輝点を呈し、細菌の蛍光は、微少
なファージ粒子と区別できる大きく明るい状態を呈す
る。したがって、ファージ感染により蛍光物質で標識さ
れたファージDNAが注入され染色された細菌(つまり
特定の生細菌)は、この蛍光物質を媒介として短時間の
うちに光学的に容易に検出できる。この利点は、蛍光物
質に限らず標識物質として色素を用いた場合も同様も得
られる。The invention according to this proposal has the following features.
That is, a phage that is a bacterial virus specifically binds to a host bacterium, injects phage DNA into living cells only depending on the membrane potential of the bacterium, and injects the phage DNA into the cells. If the phage DNA is labeled with a dye or a fluorescent substance, the whole host bacterium is stained to exhibit a large bright spot, because the phage DNA has a property of spreading (developing) in a spatially wide cell. Bacterial fluorescence exhibits a large, bright state that can be distinguished from tiny phage particles. Therefore, a bacterium (ie, a specific living bacterium) into which phage DNA labeled with a fluorescent substance has been injected and stained by phage infection can be optically easily detected in a short time by using the fluorescent substance. This advantage can be obtained not only for the fluorescent substance but also when a dye is used as the labeling substance.
【0016】そしてこの提案の発明は、上述したレポー
タ遺伝子において用いる遺伝子組換え法のようにファー
ジ毎(つまり細菌と特異的な関係にあるファージ毎)に
特殊なファージDNAをDNA組換え操作で調製する必
要がなく、基本的には共通した同一、単一の普遍性のあ
る操作を用いてファージDNAを標識できる点で優れて
いる。According to the proposed invention, a special phage DNA is prepared by a DNA recombination operation for each phage (that is, for each phage having a specific relationship with a bacterium), as in the above-described gene recombination method for the reporter gene. This is advantageous in that phage DNA can be labeled using a common, identical and single universal operation.
【0017】[0017]
【発明が解決しようとする課題】ところで、上記特願平
9−240919号の方法による目的細菌の検出は、例
えば、測定対象の試料液に核酸(DNA)を標識したフ
ァージを一定量添加して宿主細菌と接触させて染色し、
これを検出器に導入し、細菌から発せられるシグナル
(色や蛍光)を検出する手順で行なうことができるが、
本発明者のその後の研究によれば、この方法を実際の工
業的なレベルで実施するためには更に以下のような問題
のあることが知見された。The detection of the target bacterium by the method of Japanese Patent Application No. 9-240919 is performed, for example, by adding a certain amount of phage labeled with nucleic acid (DNA) to a sample solution to be measured. Stain by contacting with host bacteria,
This can be done by introducing this into a detector and detecting the signal (color or fluorescence) emitted from the bacteria.
According to subsequent studies by the present inventors, it has been found that the following problems are further encountered in implementing this method at an actual industrial level.
【0018】すなわち、測定試料中に細菌に感染しなか
ったフリーの標識ファージが多量に残存するために、こ
のフリーのファージに含まれる標識物質から発せられる
シグナル(色や蛍光)が上述のバックグラウンドノイズ
となって、検出感度の低下を招くという問題がある。す
なわち、試料中の細菌を迅速に染色するためには、細菌
濃度の高低如何によらず、添加するファージ溶液の濃度
は一定値以上(一般的には1×1010個/1ml以上)
であることが必要と考えられるが、このような高濃度の
標識ファージを含む試料液は、そのバックグラウンドの
輝度が高くなり、染色した細菌を明瞭な点として検出す
ることが難しくなる。このことは後述する参考例の結果
により実証的に確認される。That is, since a large amount of free labeled phage that has not been infected with bacteria remains in the measurement sample, the signal (color or fluorescence) emitted from the labeled substance contained in the free phage is the above-mentioned background. There is a problem that noise is caused and the detection sensitivity is reduced. That is, in order to rapidly stain the bacteria in the sample, the concentration of the phage solution to be added is not less than a certain value (generally, not less than 1 × 10 10 cells / ml), regardless of the bacterial concentration.
However, the sample solution containing such a high concentration of labeled phage has a high background luminance, and it is difficult to detect stained bacteria as clear points. This is empirically confirmed by the results of Reference Examples described later.
【0019】そしてこのフリーの標識ファージ等による
バックグランドノイズが高くなるという問題は、細菌や
ファージの濃度それ自体の影響がある他、これらの相対
的な濃度関係にも影響されるため問題は複雑である。The problem of high background noise caused by free labeled phage and the like is complicated not only by the concentration of bacteria and phage itself but also by the relative concentration relationship between them. It is.
【0020】本発明者は以上のような種々の問題を検討
し、実際の工業的な段階で実施するに当たっては、より
具体的な実施化段階での工夫が求められるという観点か
ら更に研究・開発を進め、本発明をなすに至ったのであ
る。The present inventor has studied the various problems described above, and further studies and developments from the viewpoint that, when implementing the present invention at the actual industrial stage, it is required to devise a more concrete implementation stage. And led to the present invention.
【0021】[0021]
【課題を解決するための手段及び作用】上記の目的を達
成するために、本願は前記した特許請求の範囲の各請求
項に記載した発明を提供するものであり、これは、目的
とする宿主細菌は捕捉するが、細菌より微細な夾雑物や
フリーのファージは透過するようなろ過材を選択し、こ
れによって測定対象の試料液をろ過させ、フリーの標識
ファージや夾雑物由来のバックグラウンドノイズを除去
することで、感度向上を図るものである。In order to achieve the above object, the present application provides the invention described in each of the claims set forth above. Select a filter material that captures bacteria but allows finer impurities and free phage to penetrate than bacteria, thereby filtering the sample solution to be measured, and free labeled phage and background noise derived from the impurities. Is removed to improve the sensitivity.
【0022】上記目的の達成のために提案する本願請求
項1の細菌の検出方法の発明は、色素又は蛍光物質で核
酸(DNA)を標識したファージを宿主細菌に接触させ
ることで該標識DNAを宿主細菌に注入して細菌を染色
し、この染色した状態を光学的に検出する方法におい
て、測定対象の細菌を含む試料液をろ過材でろ過するこ
とで、該試料液中の細菌をろ過材で捕捉すると共に、試
料液に含まれる色素,蛍光物質を除去し、その後、宿主
細菌に特異的でかつDNAを標識したファージをろ過材
上に捕捉した細菌に接触させて上記染色を行わせ、この
染色した細菌を計数することを特徴とする。[0022] The invention of the method for detecting bacteria according to claim 1 of the present invention, which is proposed to achieve the above object, comprises the steps of: contacting a phage labeled with a nucleic acid (DNA) with a dye or a fluorescent substance to a host bacterium; In a method of injecting a host bacterium to stain the bacterium and optically detecting the stained state, the bacteria in the sample fluid are filtered through a filter material by filtering a sample solution containing the bacterium to be measured. And removing the dye and fluorescent substance contained in the sample solution, and then contacting a phage specific to the host bacterium and labeled with DNA to the bacterium captured on the filter medium to perform the staining, The stained bacteria are counted.
【0023】また、本願請求項3の発明は、色素又は蛍
光物質でDNAを標識したファージを宿主細菌に接触さ
せることで該標識DNAを宿主細菌に注入して細菌を染
色し、この染色した状態を光学的に検出する細菌の方法
において、宿主細菌に特異的でかつDNAを標識したバ
クテリオファージを含むファージ溶液を測定対象の細菌
を含む試料液に添加して細菌を染色し、この溶液をろ過
材でろ過して上記染色した細菌を捕捉すると共に、該溶
液に含まれる色素,蛍光物質を除去した後、染色した細
菌を計数することを特徴とする。[0023] The invention of claim 3 of the present application relates to a method of contacting a phage labeled with DNA with a dye or a fluorescent substance to a host bacterium, injecting the labeled DNA into the host bacterium, and staining the bacterium. In a bacterial method for optically detecting a bacterium, a phage solution containing a bacteriophage specific to a host bacterium and labeled with DNA is added to a sample solution containing the bacterium to be measured, and the bacterium is stained. The method is characterized in that the stained bacteria are captured by filtering through a material, the dye and the fluorescent substance contained in the solution are removed, and then the stained bacteria are counted.
【0024】これらの発明において用いる「ろ過材」と
しては、細菌は通過しない孔径の範囲で出来るだけ大き
な孔径の通液開孔をもつろ過膜、例えば、精密ろ過膜
(マイクロフィルタ:MF),限外ろ過膜等が好ましく
用いられる。As the "filtering material" used in these inventions, a filtration membrane having a pore opening as large as possible within the range of pores through which bacteria do not pass, such as a microfiltration membrane (microfilter: MF), An outer filtration membrane or the like is preferably used.
【0025】また、ろ過材に捕捉した細菌は、回収液に
分離回収してから細菌数として計数することができる。
このためのろ過材としては、細菌の吸着し難い、親和性
に乏しい素材で構成されているろ過材(セルロースアセ
テート製,ポリカーボネート製)を好ましく用いること
ができる。Further, the bacteria captured by the filter medium can be counted as the number of bacteria after being separated and recovered in a recovery solution.
As a filter material for this purpose, a filter material (made of cellulose acetate or polycarbonate) made of a material which is hardly adsorbed by bacteria and has poor affinity can be preferably used.
【0026】また上記の発明においてファージDNAに
標識する色素としては、例えばエチジウムブロミド,プ
ロピジウムイオデートなどを例示でき、蛍光物質として
はアクリジンオレンジ(C17H20ClN3 :以下「AC
OR」と略記する)、4,6−ジアミジン−2−フェニ
ルインドールハイドロクロライド(DAPI)等を例示
することができる。In the above-mentioned invention, examples of the dye for labeling the phage DNA include ethidium bromide and propidium iodide, and the fluorescent substance is acridine orange (C 17 H 20 ClN 3 : hereinafter “AC
OR "), 4,6-diamidine-2-phenylindole hydrochloride (DAPI) and the like.
【0027】細菌とこれに特異的なファージの組合せ
は、大腸菌とT系ファージ,λファージ等、サルモネラ
とP系ファージ等、シュードモナスとP系ファージ等、
クレブシアラとスタンフォード大学60,92の各ファ
ージ、クロストリジウムと70,71,72の各ファー
ジ、シゲラとφ80,スタンフォード大学37,D2
0、コリネバクテリウムとC系ファージ、マイクロコッ
カスとN系ファージ,ML53−40 ファージなどを
例示的に挙げることができる。Combinations of bacteria and phages specific thereto include Escherichia coli and T-type phages, λ phages, etc., Salmonella and P-type phages, Pseudomonas and P-type phages, etc.
Klebsiara and each phage of Stanford University 60,92, Clostridium and each phage of 70,71,72, Shigella and φ80, Stanford University 37, D2
0, Corynebacterium and C-type phage, Micrococcus and N-type phage, ML53-40 phage, and the like.
【0028】また、ろ過材に捕捉されている染色細菌を
少量の回収液に分離回収し、これを既知の投光−受光系
の光学的検出手段を付帯したフローセルに流して、通過
する輝点の.光強度を予め定めた閾値と比較し、閾値を
越えた輝点の数をカウントする既知のフローセル系検出
装置を用いて、オシロスーコプに表示する等、細菌の計
数を行なうことができる。例えば標識物質として上記A
CORを使用する場合には、投光系の励起光源として渡
長488nmのレーザー光源を使用し、受光系において
510〜560nm付近の蛍光を検出することができ
る。Further, the stained bacteria trapped in the filter medium is separated and collected into a small amount of a collecting solution, and the separated bacteria are passed through a flow cell equipped with a known light-receiving / light-receiving system optical detecting means, and the bright spot passing therethrough. of. Bacteria can be counted, for example, displayed on an oscilloscope, using a known flow cell detection device that compares the light intensity with a predetermined threshold and counts the number of bright spots exceeding the threshold. For example, as the labeling substance A
When COR is used, a laser light source having a wavelength of 488 nm is used as an excitation light source of the light projecting system, and fluorescence in the vicinity of 510 to 560 nm can be detected in the light receiving system.
【0029】細菌よりも小さい蛍光物質等は、試料液全
体の背景(以下「バックグラウンド」という)の光強度
レベルを高くし、その結果として、光強度を目安として
細菌検出を行う場合には、染色細菌とバックグラウンド
の差が小さくなって検出を難しくする傾向を招くが、本
発明によれば、これらの誤検出等の原因となる試料液由
来の色素,蛍光物質は除去されるので、感度のよい細菌
検出が実現できる。When a fluorescent substance or the like smaller than bacteria increases the light intensity level of the background (hereinafter referred to as "background") of the entire sample solution, as a result, when bacteria are detected based on the light intensity, Although the difference between the stained bacterium and the background becomes small and the detection tends to be difficult, according to the present invention, the dye and the fluorescent substance derived from the sample solution, which cause these erroneous detections, are removed. Good bacterial detection can be realized.
【0030】発明において「ろ過材でろ過して除去すべ
き色素,蛍光物質等」としては、フリーの標識ファージ
の他、例えば測定対象の試料液が食品排水、パルプ排
水、界面活性剤を含む河川水等に含まれる色素顔料,漂
白剤,食品添加物等の色素あるいは蛍光物質が試料液由
来として含まれる可能性がある。また、蛍光物質で標識
したファージ液を例えば常温(通常は冷蔵保存)で3ヶ
月放置した後に蛍光顕微鏡で観察すると、ファージの微
細な蛍光の他に、放置前には観察されていなかった種々
の大きさの蛍光を発する粒子等が観察されることがあ
る。これは、ファージが劣化して内部のDNAが露出し
これらが絡みあって大きな凝集物を作って、蛍光性の粒
子(夾雑物)となって蛍光を発する点になることを推定
させる。In the present invention, examples of the "dye, fluorescent substance, etc. to be removed by filtration with a filter medium" include, in addition to free labeled phage, for example, a sample liquid to be measured contains food wastewater, pulp wastewater, and a river containing surfactant. There is a possibility that a pigment or a fluorescent substance such as a pigment, a bleach, or a food additive contained in water or the like may be contained as a sample liquid. Further, when a phage solution labeled with a fluorescent substance is allowed to stand at room temperature (usually refrigerated) for 3 months, for example, and observed with a fluorescence microscope, it is found that in addition to the fine fluorescence of the phage, various phages not observed before the phage were left. Particles or the like that emit fluorescent light of a size may be observed. This suggests that the phage is degraded, the DNA inside is exposed, and these are entangled to form large aggregates, which become fluorescent particles (contaminants) and emit fluorescence.
【0031】本願請求項2の発明は、測定対象の細菌を
含む試料液をろ過材でろ過することで、該試料液中の細
菌をろ過材で捕捉すると共に、試料液に含まれる色素,
蛍光物質を除去し、その後、このろ過材に対し試料液の
通液ろ過方向とは反対方向に宿主細菌に特異的でかつD
NAを標識したファージを含むファージ液を通液ろ過し
てファージを細菌に接触させることで染色を行わせてい
るため、ファージの劣化による凝集物(蛍光性の粒子)
はろ過材の上面には通過せず、凝集していない正常なフ
ァージのみが上面に通過し、細菌と接触するため、凝集
物が誤検出の原因とならず、感度のよい細菌検出が行え
る。According to the invention of claim 2 of the present application, by filtering a sample solution containing bacteria to be measured with a filter medium, the bacteria in the sample solution are captured by the filter medium, and the dye contained in the sample solution is removed.
The fluorescent substance is removed, and then the filter medium is specific to the host bacterium in a direction opposite to the direction of filtration of the sample solution and has a D value.
Aggregates due to phage degradation (fluorescent particles) because the phage is stained by passing through a phage solution containing NA-labeled phage and filtering the phage into contact with bacteria.
Does not pass through the upper surface of the filter medium, and only normal phages that have not aggregated pass through the upper surface and come into contact with bacteria, so that aggregates do not cause erroneous detection, and highly sensitive bacteria can be detected.
【0032】ろ過材に捕捉した細菌をそのまま検出する
装置としては、膜上に捕捉された細菌(標識ファージに
より染色された細菌)をそのまま導入する以下の装置、
例えば、顕微鏡等の光学的検出手段で目視で計数する
か、あるいは標識物質として蛍光色素を用いた場合、膜
に励起光を照射して膜上の細菌が発した蛍光を対物レン
ズで集光し、この光をCCDカメラ等により撮像して電
気信号に変換し、画像処理装置(コンピュータ)で、予
め定めた大きさ以上の輝点のもの、あるいは光強度が予
め定めた閾値以上の輝点を細菌と判定することができ
る。発色色素の場合も同様に、膜表面画像をCCDカメ
ラ等で捕らえ、標識物質が発する色をもつ輝点につい
て、規定値以上の大きさや発色強度が見られたものを細
歯と判定することができる。As the apparatus for directly detecting the bacteria captured on the filter medium, the following apparatus for directly introducing the bacteria captured on the membrane (the bacteria stained by the labeled phage),
For example, counting is performed visually with an optical detection means such as a microscope, or when a fluorescent dye is used as a labeling substance, the membrane is irradiated with excitation light and the fluorescence emitted by bacteria on the membrane is collected by an objective lens. This light is imaged by a CCD camera or the like and converted into an electric signal, and an image processing device (computer) uses the image processing device (computer) to generate a bright spot having a predetermined size or more or a bright spot having a light intensity equal to or more than a predetermined threshold. Bacteria can be determined. Similarly, in the case of coloring pigments, a film surface image is captured by a CCD camera or the like, and a bright spot having a color emitted by the labeling substance can be determined as a fine tooth if the size or color intensity is greater than a specified value. it can.
【0033】[0033]
【発明の実施の形態】以下、本発明をより具体的な実施
の態様に基づいて説明する。DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described based on more specific embodiments.
【0034】上記発明において、ろ過材としては、代表
的にはろ過膜を好ましく用いることができ、その用い方
としてより好適には、試料液の通液ろ過により該試料液
に含まれる細菌の捕捉と、同じくこれに含まれる色素,
蛍光物質の除去とを行なった後、このろ過膜に対し試料
液の通液ろ過方向とは反対方向に上記DNA(核酸)を
標識したファージを含むファージ溶液を通液ろ過するこ
とで該ファージを細菌に接触させて上記染色をする方法
を挙げることができる。なお、試料液の通液ろ過、ファ
ージ溶液の通液ろ過の後では、必要に応じて洗浄するこ
とがよい。[0034] In the above invention, typically, a filtration membrane can be preferably used as the filter medium, and more preferably, the capture of bacteria contained in the sample solution by filtration of the sample solution is performed. And the pigments contained therein,
After removing the fluorescent substance, the phage is filtered through a phage solution containing the phage labeled with the DNA (nucleic acid) in a direction opposite to the direction of the filtration of the sample solution through the filtration membrane. A method of performing the above-mentioned staining by contacting with bacteria can be mentioned. After the filtration of the sample solution and the filtration of the phage solution, washing may be performed as necessary.
【0035】このようにすることで、ファージ溶液に含
まれることがある培地成分等をろ過膜でろ過除去でき、
誤検出の虞れをより一層低減することができる。By doing so, the medium components and the like that may be contained in the phage solution can be removed by filtration through the filtration membrane.
The possibility of erroneous detection can be further reduced.
【0036】また上記発明のように、ろ過材に捕捉後の
細菌にファージを接触させて染色することに代えて、測
定対象の細菌を含む試料液に、DNA(核酸)を標識し
たファージを含むファージ溶液を添加することで上記染
色をし、その後、この溶液をろ過膜等のろ過材でろ過す
ることで上記染色した細菌の捕捉と該溶液に含まれる色
素,蛍光物質の除去とを行なうこともできる。As described above, instead of contacting the phage with the bacteria captured by the filter medium and staining the phage, the sample solution containing the bacteria to be measured contains phages labeled with DNA (nucleic acid). The above-mentioned staining is performed by adding a phage solution, and thereafter, the solution is filtered through a filtering material such as a filtration membrane to capture the stained bacteria and remove the dyes and fluorescent substances contained in the solution. Can also.
【0037】これらの方法によりろ過することで、フリ
ーの標識ファージを除去することができるので、バック
グラウンドノイズを低減させて、検出感度を向上させる
ことができる。By filtering by these methods, free labeled phage can be removed, so that background noise can be reduced and detection sensitivity can be improved.
【0038】また、ろ過材に捕捉されている染色細菌
は、回収液でろ過材を逆洗し、あるいは回収液に浸漬す
ることで、ろ過材から回収液に分離回収することができ
る。The stained bacteria captured by the filter medium can be separated and collected from the filter medium by backwashing the filter medium with the recovery liquid or immersing the filter medium in the recovery liquid.
【0039】これらの方法によれば、測定対象の試料液
をろ過して宿主細菌のみを捕捉することで、試料中には
じめから含まれる微細な色素,蛍光物質等の夾雑物を除
去することができる。なお、かかる夾雑物を除去した細
菌を染色してこれを検出するには、以下のようないくつ
かの手順が考えられる。According to these methods, by filtering the sample solution to be measured and capturing only the host bacteria, it is possible to remove impurities such as fine pigments and fluorescent substances originally contained in the sample. it can. In order to stain and detect the bacteria from which such contaminants have been removed, the following several procedures can be considered.
【0040】例えば、宿主細菌を捕捉したろ過材に直接
標識ファージ液を添加接触させて、ろ過材表面で宿主細
菌と標識ファージを接触させた後、さらに洗浄液を通液
して、フリーのファージを除去する。その後はろ過材を
そのまま検出装置に導入して染色細菌数を計数してもよ
いし、あるいは染色した細菌をろ過材から回収液中に剥
離して回収し、フロー系検出装置に導入することもでき
る。For example, the labeled phage solution is directly added to and contacted with the filter medium capturing the host bacteria, and the host bacteria and the labeled phage are brought into contact on the surface of the filter medium. Remove. Thereafter, the filter media may be directly introduced into the detection device to count the number of stained bacteria, or the stained bacteria may be separated from the filter media into the recovery solution and collected, and then introduced into the flow detection device. it can.
【0041】さらに、保存されている標識ファージ溶液
には、外界から浮遊物や細菌が混入して、大きな夾雑物
が含まれる可能性があって、これらがまれに色や蛍光を
発する虞れが考えられる場合には、請求項2の発明のよ
うに、ろ過材としてろ過膜を用い、測定試料液のろ過方
向とは逆の方向(つまり膜の裏側から逆洗する操作)か
ら標識ファージ溶液を通液ろ過すれば、ファージは膜を
透過して膜の表側に捕捉された菌と反応するが、大きな
夾雑物は透過できずに膜の裏側に捕捉されるため、ファ
ージだけを宿主細菌に確実に接触させるという方法をと
ることができる。さらにこの方法の利点は、試料液をろ
過したことで、ろ過材表面に吸着してしまった宿主細菌
を剥がして少量の回収液中に浮遊させることで、ファー
ジとの接触効率を良くするという効果もある。この発明
のために好適に用いることができるろ過材としては、フ
ァージの通過を許容する範囲でそれ以上の大きさの物質
の通過を阻止するできるだけ小さな孔径のろ過膜を用い
ることがよい。Furthermore, the stored labeled phage solution may contain large contaminants due to the incorporation of suspended matters and bacteria from the outside world, which may rarely emit color or fluorescence. If it is conceivable, as in the second aspect of the present invention, the labeled phage solution is used in a direction opposite to the filtration direction of the measurement sample solution (that is, an operation of backwashing from the back side of the membrane) using a filtration membrane as a filtration material. When filtration is performed, the phage penetrates the membrane and reacts with the bacteria trapped on the front side of the membrane.However, large contaminants cannot be permeated and are trapped on the back side of the membrane. Can be brought into contact with the object. Another advantage of this method is that, by filtering the sample solution, the host bacteria adsorbed on the surface of the filter material are peeled off and suspended in a small amount of recovered solution, thereby improving the contact efficiency with phage. There is also. As a filter medium that can be suitably used for the present invention, it is preferable to use a filter membrane having a pore size as small as possible that blocks the passage of a substance having a size larger than the allowable range of phage passage.
【0042】この請求項2の発明によれば、標識ファー
ジ添加後にろ過するため、夾雑物のみならず、同時にフ
リーの標識ファージをも除去するのでバックグラウンド
ノイズを低減できるという利点があり、特に細菌の低濃
度試料液についての検出に有効である。According to the second aspect of the present invention, since the filtration is performed after the addition of the labeled phage, not only the contaminants but also the free labeled phage are removed at the same time, so that there is an advantage that the background noise can be reduced. Is effective for detecting low concentration sample solutions.
【0043】すなわち、宿主細菌と標識ファージを確実
に接触させ、細菌とファージが反応して染色が行われる
ためには、宿主細菌の濃度に関わらず、標識ファージを
ある一定量以上の濃度になるように添加する必要があ
り、例えば、大腸菌とファージT4を用いた系で、標識
物質としてACORを使用した場合、既知濃度の大腸菌
を含む培養液に標識ファージを添加したところ、大腸菌
をすべて染色するためには、菌濃度に関わりなく、工業
的なレベルでの迅速な検出を可能とするためには標識フ
ァージを1×1010個/ml以上の濃度になるように添
加するのが望ましいことが確認されているからである。
これは、ファージ溶液中の標識ファージが細菌に接触す
る可能性は主に拡散に律速されるからである。従って、
標識ファージの添加量が試料液の体積に比例するため、
標識ファージの使用量をできるかぎり削減するために
は、ろ過材上に細菌を捕捉するという濃縮操作の後にフ
ァージ溶液を添加する方法が好ましく用いられるのであ
る。That is, to ensure that the host bacterium is brought into contact with the labeled phage and that the bacterium reacts with the phage for staining, the concentration of the labeled phage becomes a certain level or more regardless of the concentration of the host bacterium. For example, when ACOR is used as a labeling substance in a system using Escherichia coli and phage T4, when labeled phages are added to a culture solution containing Escherichia coli at a known concentration, all Escherichia coli is stained. Therefore, it is desirable to add labeled phage to a concentration of 1 × 10 10 cells / ml or more to enable rapid detection at an industrial level regardless of the bacterial concentration. This is because it has been confirmed.
This is because the probability that the labeled phage in the phage solution comes into contact with the bacteria is mainly governed by diffusion. Therefore,
Since the amount of labeled phage added is proportional to the volume of the sample solution,
In order to reduce the usage of labeled phage as much as possible, a method of adding a phage solution after a concentration operation of capturing bacteria on a filter medium is preferably used.
【0044】また、菌の低濃度試料液に含まれる細菌
を、その数分の1乃至数十分の1以下の回収液に分離回
収するので、フロー系検出装置に流す液量を少量化で
き、迅速な検出を実現でき、これらの効果は、試料液5
0ml以上に対して回収液の液量を10ml以下、好ま
しくは1ml以下に少なくする場合に特徴的に得られ
る。In addition, the bacteria contained in the low concentration sample solution of bacteria are separated and recovered into one-several to several tenths of the recovering liquid, so that the amount of the liquid flowing to the flow detecting device can be reduced. , Quick detection can be realized, and these effects are
It is characteristically obtained when the amount of the recovered liquid is reduced to 10 ml or less, preferably 1 ml or less with respect to 0 ml or more.
【0045】上記のフロー系検出装置には、測定対象の
試料液に励起光が照射されることにより標識物質が発す
る蛍光を、この試料液がフローセル内を通過する際に対
物レンズにより集光し、その光信号を光電子増倍管で受
光して電気信号に変換し、増幅等の電気的な信号処理を
行なって、オシロスコープにパルス信号として出力する
ようにしたものを用いることができる。In the above-mentioned flow system detection apparatus, the fluorescence emitted from the labeling substance when the sample liquid to be measured is irradiated with the excitation light is condensed by the objective lens when the sample liquid passes through the flow cell. Alternatively, the optical signal may be received by a photomultiplier, converted into an electric signal, subjected to electric signal processing such as amplification, and output as a pulse signal to an oscilloscope.
【0046】[0046]
【実施例】以下に、本発明の実施例を述べる。Embodiments of the present invention will be described below.
【0047】標識物質としてACOR(アクリジンオレ
ンジ)を使用した。このACORはDNAにインターカ
レーション結合することで、波長490nm付近の励起
光照射により、波長500〜600nm付近の蛍光を発
する蛍光物質である(特願平10−130324号、特
願平10−130325号参照)。ACOR (acridine orange) was used as a labeling substance. The ACOR is a fluorescent substance that emits fluorescence at a wavelength of about 500 to 600 nm upon irradiation with excitation light at a wavelength of about 490 nm by intercalation bonding with DNA (Japanese Patent Application Nos. 10-130324 and 10-130325). No.).
【0048】ACORの発する蛍光を検出するために、
投光系は励起光源として波長488nmのレーザー光源
を使用し、受光系は510〜560nm付近の蛍光を検
出するフロー系検出装置を用いた。To detect the fluorescence emitted by ACOR,
For the light projecting system, a laser light source having a wavelength of 488 nm was used as an excitation light source, and for the light receiving system, a flow system detecting device for detecting fluorescence around 510 to 560 nm was used.
【0049】参考例1:フリーの標識ファージによるバ
ックグラウンドノイズの検討 大腸菌を含む培養液(以下「培養原液」という)に、A
CORで標識したT4ファージ(以下「ACORファー
ジ」という)を5×1010個/mlになるように添加し
て、大腸菌を染色して試料を調製した。Reference Example 1: Examination of background noise by free labeled phage A culture solution containing Escherichia coli (hereinafter referred to as "culture stock solution")
T4 phage labeled with COR (hereinafter referred to as “ACOR phage”) was added at 5 × 10 10 cells / ml, and E. coli was stained to prepare a sample.
【0050】この試料を蛍光顕微鏡で観察したところ、
大腸菌は緑色に染色されており、その周りに標識ファー
ジ由来の示さな蛍光粒子が観察できた。When this sample was observed with a fluorescence microscope,
Escherichia coli was stained green, and marked fluorescent particles derived from the labeled phage could be observed around it.
【0051】次に、この試料0.5mlをそのまま上述
したフロー系検出装置に導入したところ、バックグラウ
ンドノイズが大きく、大腸菌のシグナルがノイズに埋も
れてしまい検出できなかった。Next, when 0.5 ml of this sample was directly introduced into the above-mentioned flow detection apparatus, the background noise was large, and the E. coli signal was buried in the noise and could not be detected.
【0052】そこで、この試料を生理食塩水で10,0
00倍希釈して、再度、フロー系検出装置に導入したと
ころ、バックグラウンドノイズは低下し、染色された大
腸菌から発せられた蛍光と判断できるパルス信号が検出
されたので、このパルス信号の数をカウントして、培養
原液の菌濃度を算出した。Therefore, this sample was dissolved in physiological saline for 10.0 or less.
When diluted 100-fold and introduced into the flow detection device again, the background noise was reduced, and a pulse signal that could be judged as the fluorescence emitted from the stained Escherichia coli was detected. By counting, the bacterial concentration of the undiluted culture solution was calculated.
【0053】一方、既知のコロニーカウント法により培
養原液の菌濃度を求めた。すなわち、同じ大腸菌を含む
培養原液の一部を、大腸菌が増殖できるLB寒天培地に
塗沫し、37℃で一晩静置後、生じたコロニー数を計測
して、該培養原液の菌濃度を算出した。On the other hand, the bacterial concentration of the undiluted culture solution was determined by a known colony counting method. That is, a part of the culture stock containing the same Escherichia coli is spread on an LB agar medium in which Escherichia coli can grow, left at 37 ° C. overnight, the number of colonies formed is counted, and the bacterial concentration of the culture stock is determined. Calculated.
【0054】これらのフロー系検出装置により検出した
培養原液の菌濃度、およびコロニーカウント法により得
た培養原液の菌濃度は、ともに概ね1.29×108個
/mlとなり、良く一致した。The bacterial concentration of the undiluted culture solution detected by these flow detection devices and the bacterial concentration of the undiluted culture solution obtained by the colony counting method were approximately 1.29 × 10 8 cells / ml, which were in good agreement.
【0055】これらの結果から、細菌を染色するのに必
要と考えられる標識ファージの濃度では、蛍光顕微鏡等
を用いた目視による検出は可能であるが、フロー系検出
装置を用いる場合には、フリーの標識ファージ由来の蛍
光によるバックグラウンド、に染色細菌の蛍光が埋もれ
てしまい、感度のよい検出ができないことが分かる。ま
た、希釈によってバックグラウンドの蛍光を低減すれ
ば、フロー系検出装置を用いた検出方法により、細菌数
の計測が可能であることが分かる。From these results, it is possible to visually detect the concentration of labeled phage, which is considered necessary for staining bacteria, using a fluorescence microscope or the like. It can be seen that the fluorescence of the stained bacteria was buried in the background due to the fluorescence derived from the labeled phage, and that highly sensitive detection was not possible. Also, if the background fluorescence is reduced by dilution, it can be seen that the number of bacteria can be measured by a detection method using a flow detection device.
【0056】参考例2:菌数が低濃度のときの測定不良 上記大腸菌の培養原液をあらかじめ生理食塩水で10,
000倍希釈したものを原液とした。参考例lのコロニ
ーカウントの結果より、この原液の菌濃度は1.29×
104個/mlである。Reference Example 2: Insufficient measurement when the number of bacteria is at a low concentration.
A 000-fold dilution was used as a stock solution. From the results of the colony count of Reference Example 1, the bacterial concentration of this stock solution was 1.29 ×
10 4 cells / ml.
【0057】この原液にACORファージを5×1010
個/mlになるように添加して、大腸菌を染色した。こ
れを、そのままフロー系検出装置に導入したところ、バ
ックグラウンドノイズが大きくなり検出できなかった。To this stock solution, 5 × 10 10 ACOR phage were added.
E. coli was stained by adding so that the number of cells per ml was increased. When this was directly introduced into a flow system detection device, the background noise became large and could not be detected.
【0058】そこで、フリーの標識ファージ由来の蛍光
によるバックグラウンドを低減するために10,000
倍希釈し、これをフロー系検出装置に導入したところ、
バックグラウンドは低減されたが、染色された大腸菌か
ら発せられたと判断できるパルス信号も検出できなかっ
た。Therefore, in order to reduce the background due to the fluorescence derived from the free labeled phage, 10,000
When it was diluted by a factor of two and introduced into a flow detection device,
Although the background was reduced, no pulse signal that could be judged to be emitted from stained E. coli was detected.
【0059】この結果から、希釈によるバックグラウン
ドの低減は、参考例1のような菌濃度の高い試料ならば
問題はないが、濃度が低い参考例2のような試料は正確
な計測ができなくなることが分かる。From the results, it is clear that the background can be reduced by dilution if the sample has a high bacterial concentration as in Reference Example 1, but the sample as in Reference Example 2 with low concentration cannot be measured accurately. You can see that.
【0060】参考例3:ファージ溶液中の誤差原因粒子 常温で3カ月放置したファージ液(1×1011個/m
l)を直接フロー系検出装置に導入し、細菌と同等のパ
ルス信号を検出して、その濃度を算出したところ、5×
105個/mlという結果が得られた。つまり、このフ
ァージ液中にはDNAの凝集などにより生じた菌と同等
の大きさの蛍光性夾雑物が5×105個/mlの濃度で
存在していると推定される。このファージ液を用いて以
下の実験を行った。 (1) (大腸菌培養液(菌濃度約1×105個/ml)5ml
をフィルタろ過) ↓ (膜上部より、上記ファージ液1mlをろ過し、フィル
タ表面で菌とファージを反応させる) ↓ (洗浄液として生理食塩水10mlをフィルタ上部より
ろ過し、余分なファージを除去) ↓ (逆洗液として生理食塩水5mlをフィルタの反対側か
らろ過し、この透過液を回収) ↓ (回収した逆洗液をフロー系検出装置に導入し、染色さ
れた菌のパルス数をカウントして、逆洗液の菌濃度を算
出したところ、2.1×105個/mlとなった)。 (2) (大腸菌培養液(菌濃度1×105個/ml)5mlを
フィルタろ過) ↓ (膜上側に1mlのシリンジを挿したまま、ACORフ
アージ濃度5×1010個/mlのファージ液1mlをフ
ィルタ下部よりろ過し、フィルタ表面付近で菌とファー
ジを反応させる) ↓ (洗浄液として生理食塩水10mlをフィルタ上部より
ろ過し、余分なファージを除去) ↓ (逆洗液として生理食塩水5mlをフィルタの反対側か
らろ過し、この透過液を回収) ↓ (回収した逆洗液をフロー系検出装置に導入し、染色さ
れた菌のパルス数をカウント)。Reference Example 3: Particles causing error in the phage solution The phage solution (1 × 10 11 cells / m 3) left at room temperature for 3 months
l) was directly introduced into a flow detection device, and a pulse signal equivalent to that of a bacterium was detected.
Results of 10 5 cells / ml was obtained. In other words, it is presumed that a fluorescent contaminant having a size equivalent to that of bacteria produced by aggregation of DNA or the like is present in the phage solution at a concentration of 5 × 10 5 cells / ml. The following experiment was performed using this phage solution. (1) 5 ml of Escherichia coli culture solution (concentration about 1 × 10 5 cells / ml)
↓ (1 ml of the above phage solution is filtered from the upper part of the membrane, and bacteria and phage are reacted on the filter surface) ↓ (10 ml of physiological saline as a washing liquid is filtered from the upper part of the filter to remove excess phage) ↓ (5 ml of physiological saline is filtered from the opposite side of the filter as the backwashing solution, and the permeate is collected.) ↓ (The collected backwashing solution is introduced into the flow detection device, and the number of stained bacterial pulses is counted. The bacterial concentration of the backwash was calculated to be 2.1 × 10 5 cells / ml). (2) (Filter filtration of 5 ml of Escherichia coli culture solution (concentration of 1 × 10 5 cells / ml)) ↓ (1 ml of a phage solution having an ACOR phage concentration of 5 × 10 10 cells / ml with a 1 ml syringe inserted on the upper side of the membrane) Is filtered from the lower part of the filter, and the bacteria and phage are reacted in the vicinity of the filter surface. ↓ (10 ml of physiological saline as a washing liquid is filtered from the upper part of the filter to remove excess phage) ↓ (5 ml of physiological saline as a back washing liquid) Filter from the opposite side of the filter and collect the permeate. ↓ (Introduce the collected backwash into the flow detection device and count the number of pulses of stained bacteria).
【0061】以上によりカウントされた逆洗液中の菌濃
度は、1.1×105個/mlとなった。The bacterial concentration in the backwash solution counted as described above was 1.1 × 10 5 cells / ml.
【0062】これらの(1),(2)の結果から、
(1)では、ファージ液中の蛍光性夾雑物が菌数に上乗
せされて検出されたのに対して、(2)では、ファージ
液を菌が捕捉された膜面とは反対の側からろ過すること
で、サンプル側に蛍光性夾雑物が混入することが避けら
れ、菌のみを検出できることが確認された。From the results of (1) and (2),
In (1), fluorescent contaminants in the phage solution were detected by being added to the number of bacteria, whereas in (2), the phage solution was filtered from the side opposite to the membrane surface on which the bacteria were captured. By doing so, it was confirmed that fluorescent contaminants were prevented from being mixed into the sample side, and only bacteria could be detected.
【0063】参考例4:ろ過膜を用いた細菌の捕捉と分
離回収 下記のろ過膜を用いて、上記参考例2の菌原液(希釈後
のもので、菌濃度1.29×104個/ml)をろ過し
て、菌を膜表面に捕捉し、逆洗により回収液(生理食塩
水)中に回収してフロー系検出装置に導入する方法を、
以下の操作により行ない、回収液中への菌の回収率を調
べた。(孔径0.45μmのマイクロフィルタ(Schlei
cher & Schuell社製:FP030/2)上部より、上記
培養原液の5ml(ファージ未添加)をろ過。なお別途
この培養原液の菌濃度をコロニーカウント法により計測
した。) ↓ (洗浄液として生理食塩水10mlをフィルタ上部より
ろ過) ↓ (逆洗液として生理食塩水5mlをフィルタの反対側か
らろ過し、この透過液を回収) ↓ (逆洗液の菌濃度をコロニーカウント法により計測) ↓ (回収率の算出)。Reference Example 4: Capture and Separation and Recovery of Bacteria Using Filtration Membrane The bacterial stock solution of Reference Example 2 (after dilution, with a bacterial concentration of 1.29 × 10 4 cells / ml), the bacteria are captured on the membrane surface, collected in a recovery solution (physiological saline) by backwashing, and introduced into a flow detection device.
The following operation was performed to examine the recovery rate of the bacteria in the recovered liquid. (Micro filter with pore size 0.45μm (Schlei
5 ml of the above culture stock solution (without phage) was filtered from the top of cher & Schuell: FP030 / 2). In addition, the bacterial concentration of this culture stock solution was separately measured by a colony counting method. ↓ (10 ml of physiological saline as a washing solution is filtered from the top of the filter) ↓ (5 ml of physiological saline as a back washing solution is filtered from the opposite side of the filter and the permeate is collected) ↓ ↓ (Calculation of recovery rate).
【0064】この回収率の算出は下記式に従って行なっ
た。The recovery rate was calculated according to the following equation.
【0065】(逆洗液中の菌濃度/培養原液中の菌濃
度)×100上記により、マイクロフィルタ(MF膜)
による細菌の捕捉、生理食塩水による捕捉細菌の分離回
収の操作により、生理食塩水中への回収率70%という
結果を得た。(Bacterial concentration in backwashing solution / bacterial concentration in undiluted culture solution) × 100 Microfilter (MF membrane)
By performing the operation of capturing bacteria by the method and separating and recovering the captured bacteria by the physiological saline, a result of a recovery rate of 70% into the physiological saline was obtained.
【0066】実施例1:試料液に標識ファージを添加し
て染色後、ろ過膜で捕捉 上記参考例4の方法に従い培養原液(希釈後のもので、
菌濃度1.29×10 4個/ml)5mlにACORフ
ァージを5×1010個/mlになるように添加(つまり
使用した標識ファージの総数は2.5×1011個)し
て、大腸菌を染色させた。この試料液について、以下の
操作を行った。 (孔径0.45μmのMF膜(前出)上部より上記試料
5mlをろ過) ↓ (洗浄液として生理食塩水10mlをフィルタ上部より
ろ過) ↓ (逆洗液として生理食塩水5mlをフィルタの反対側か
らろ過し、この透過液を回収)。Example 1 A labeled phage was added to a sample solution.
After staining and capturing with a filtration membrane, a culture stock solution (after dilution,
1.29 × 10 bacteria concentration FourACOR to 5ml
5 × 10TenParts / ml (that is,
The total number of labeled phages used was 2.5 × 1011Pieces)
To stain E. coli. For this sample solution,
The operation was performed. (From the top of the MF membrane with a pore size of 0.45 µm
5ml is filtered) ↓ (10ml of physiological saline as a washing liquid from the top of the filter)
(Filtration) ↓ (5 ml of physiological saline as backwash liquid
And the permeate is collected).
【0067】上記操作を行い、回収した逆洗液をフロー
系検出装置に導入したところ、染色された大腸菌由来の
蛍光と判断できるパルス信号が確認できた。これはフリ
ーの標識ファージ由来の蛍光によるバックグラウンドノ
イズの影響はほとんどないことを意味している。つま
り、試料ろ過、および洗浄液ろ過によりフリーの標識フ
ァージはバックグラウンドに影響を及ぼさない程度まで
除去された。When the above operation was carried out and the collected backwash solution was introduced into a flow system detection apparatus, a pulse signal which could be judged as stained fluorescence derived from Escherichia coli was confirmed. This means that there is almost no influence of background noise due to fluorescence from free labeled phage. That is, free labeled phage was removed by sample filtration and washing solution filtration to such an extent that the background was not affected.
【0068】上記の菌のパルス数を計測し、原液の菌濃
度を算出したところ、0.88×104個/mlという
結果が得られた。When the number of pulses of the above bacteria was measured and the concentration of bacteria in the stock solution was calculated, a result of 0.88 × 10 4 cells / ml was obtained.
【0069】そこで、参考例4の回収率70%の結果よ
り培養原液の菌濃度を算出すると、 0.88×104(個/ml)/0.7=1.26×1
04(個/ml) となり、コロニーカウント法の結果である1.29×1
04個/mlの値とほぼ近い検出値が得られた。Therefore, when the bacterial concentration of the undiluted culture solution was calculated from the result of the recovery rate of 70% in Reference Example 4, 0.88 × 10 4 (cells / ml) /0.7=1.26×1
0 4 (cells / ml), which is the result of the colony counting method, 1.29 × 1
0 closely approximates the detected value and the value of 4 cells / ml was obtained.
【0070】実施例2:試料液ろ過後、標識ファージを
添加して染色 上記参考例2の菌原液(希釈後のもので、菌濃度は1.
29×104個/ml)500mlをフィルタ上部より
ろ過した後、以下の操作を行った。 (ACORファージ濃度5×1010個/mlのファージ
液0.5ml(つまり使用した標識ファージの総数は
2.5×1010個)をフィルタ上部よりろ過し、フィル
タ表面付近で菌とファージを反応させる) ↓ (洗浄液として生理食塩水10mlをフィルタ上部より
ろ過し、余分なファージを除去) ↓ (逆洗液(回収液)として生理食塩水5mlをフィルタ
の反対側からろ過し、この透過液を回収)。Example 2: Filtration by adding labeled phages after filtration of the sample solution and staining with the bacterial stock solution of Reference Example 2 above (after dilution, with a bacterial concentration of 1.
After filtering 500 ml of the filter (29 × 10 4 cells / ml) from the upper part of the filter, the following operation was performed. (0.5 ml of a phage solution having an ACOR phage concentration of 5 × 10 10 cells / ml (that is, the total number of labeled phages used was 2.5 × 10 10 cells) was filtered from the top of the filter, and the bacteria and phage reacted near the filter surface. ↓ (Filter 10 ml of physiological saline as a washing solution from the top of the filter to remove excess phage) ↓ (Filter 5 ml of physiological saline as a backwash solution (recovery solution) from the other side of the filter, and filter the permeate. Recovery).
【0071】回収した逆洗液をフロー系検出装置に導入
し、染色された菌のパルス数をカウントして、逆洗液
(回収液)の菌濃度を算出したところ、0.86×l0
6個/mlであり、これを回収率70%で割ると、菌濃
度は1.23×106個/mlとなった。さらに、フィ
ルタにろ過した菌原液量は500ml、逆洗液は5ml
なので、100倍濃縮されていることになる。従って、
逆洗液の菌濃度1.23×106個/mlを1/100
倍すると、菌原液の菌濃度は1.23×104個/ml
となり、コロニーカウントの結果である1.29×10
4個/mlの値に近い検出値を得た。The collected backwash solution was introduced into a flow detection device, and the number of pulses of stained bacteria was counted to calculate the bacterial concentration of the backwash solution (recovered solution).
This was 6 cells / ml, and when this was divided by a recovery rate of 70%, the bacterial concentration was 1.23 × 10 6 cells / ml. Further, the amount of the bacterial stock solution filtered through the filter is 500 ml, and the backwash solution is 5 ml.
Therefore, it is 100 times concentrated. Therefore,
Reduce the bacterial concentration of the backwash solution to 1.23 × 10 6 cells / ml by 1/100
When multiplied, the bacterial concentration of the bacterial stock solution is 1.23 × 10 4 cells / ml
And the result of colony counting is 1.29 × 10
A detection value close to the value of 4 / ml was obtained.
【0072】本例によれば、染色に要した標識ファージ
の数は、実施例1ではファージの総数2.5×1011個
であるのに対して、本例では2.5×1010個であるか
ら、10分の1量の使用で測定可能な菌の染色ができる
ことが分かる。このことから、膜ろ過を行なう本例で
は、細菌を捕捉し、染色した菌を試料液よりも少量の回
収液に分離回収してフロー系検出装置で検出するので、
標識ファージ由来のバックグランド除去効果に加えて、
試料の濃縮効果が得られ、使用する標識ファージの量を
低減することができると共に、菌が低濃度の試料を対象
として良好な測定が行なえる効果が得られる。According to the present example, the number of labeled phages required for staining was 2.5 × 10 11 in Example 1, whereas the number of labeled phages was 2.5 × 10 10 in Example 1. Therefore, it can be seen that measurable bacteria can be stained by using 1/10 of the amount. For this reason, in this example of performing membrane filtration, bacteria are captured and stained bacteria are separated and collected in a smaller amount of recovery solution than the sample solution, and detected by the flow detection device.
In addition to the background removal effect from the labeled phage,
The effect of concentrating the sample is obtained, the amount of labeled phage to be used can be reduced, and the effect that good measurement can be performed on a sample having a low concentration of bacteria is obtained.
【0073】実施例3:試料液を膜ろ過後に標識ファー
ジを膜の逆洗して染色 参考例2の菌原液(希釈後のもので、菌濃度は1.29
×104個/ml)500mlをフィルタ上部よりろ過
した後、以下の操作を行った。 (膜上側にlmlのシリンジを挿したまま、ACORフ
ァージ濃度5×1010個/mlのファージ液lmlをフ
ィルタ下部よりろ過し、フィルタ表面付近で菌とファー
ジを反応させる) ↓ (洗浄液として生理食塩水10mlをフィルタ上部より
ろ過し、余分なファージを除去) ↓ (逆洗液(回収液)として生理食塩水5mlをフィルタ
の反対側からろ過し、この透過液を回収)。Example 3: After filtering the sample solution through a membrane, the labeled phage was backwashed and stained with the membrane. The bacterial stock solution of Reference Example 2 (after dilution, with a bacterial concentration of 1.29)
(× 10 4 cells / ml) After filtering 500 ml from the upper part of the filter, the following operation was performed. (With the 1 ml syringe inserted on the upper side of the membrane, 1 ml of a phage solution having an ACOR phage concentration of 5 × 10 10 cells / ml is filtered from the lower part of the filter, and the bacteria and the phage are reacted near the filter surface.) 10 ml of water is filtered from the upper part of the filter to remove excess phage. ↓ (5 ml of physiological saline is filtered from the opposite side of the filter as a backwash liquid (recovery liquid) and the permeate is recovered).
【0074】回収した逆洗液をフロー系検出装置に導入
し、染色された菌のパルス数をカウントして、逆洗液の
菌濃度を算出したところ、0.89×106個/mlで
あり、これを回収率70%で割ると、菌濃度は1.27
×106個/mlとなった。さらに、フィルタにろ過し
た原液量は500ml、逆洗液は5mlなので100倍
濃縮されていることになる。従って、逆洗液の菌濃度
1.27×106個/mlを1/100倍すると、原液
の菌濃度は1.27×104個/mlとなった。この結
果から、実施例2の標識ファージを膜上部から添加した
場合の結果である検出値1.23×104個/mlより
も、コロニーカウントの結果である1.29×104個
/mlの値にさらに近い検出値を得ることができた。こ
の理由としては、試料をろ過した際に膜表面に吸着した
細菌が、フアージ液を逆洗ろ過することで膜から剥が
れ、溶液中に浮いた状態となり、ファージとの接触効率
が向上したためと考えられる。The collected backwash solution was introduced into a flow detection device, and the number of stained bacterial pulses was counted to calculate the bacterial concentration of the backwash solution. The result was 0.89 × 10 6 cells / ml. When this is divided by the recovery rate of 70%, the bacterial concentration is 1.27.
× 10 6 / ml. Furthermore, since the amount of the stock solution filtered through the filter is 500 ml and the backwash solution is 5 ml, it is 100 times concentrated. Therefore, when the bacterial concentration of the backwashing solution was 1.27 × 10 6 cells / ml, the bacterial concentration of the stock solution was 1.27 × 10 4 cells / ml. This result than the result detected value 1.23 × 10 4 cells / ml which is the case of adding labeled phage of Example 2 from a membrane upper, 1.29 × 10 4 cells / ml is the result of the colony count The detection value closer to the value of was obtained. The reason for this is thought to be that bacteria adsorbed on the membrane surface when the sample was filtered were peeled off from the membrane by backwashing and filtering the phage solution, and floated in the solution, improving the contact efficiency with the phage. Can be
【0075】[0075]
【発明の効果】本発明の方法によれば、簡便な操作でフ
リーの標識ファージ等の微細な色素及び/又は蛍光物質
によるバックグラウンドノイズを除去でき、目的とする
細菌の検出精度を向上させ、高感度な細菌の定量検出を
実現することができる。According to the method of the present invention, background noise due to fine dyes and / or fluorescent substances such as free labeled phages can be removed by a simple operation, and the detection accuracy of the target bacteria can be improved. Highly sensitive quantitative detection of bacteria can be realized.
【0076】特にろ過膜を用い、試料液のろ過に対し
て、ファージ溶液の通液ろ過を逆洗方向から行なうよう
にした請求項2の発明によれば、試料液に含まれるノイ
ズ成分の除去のみならず、ファージ溶液に含まれるノイ
ズ成分の除去も効果的に行なうことができて、より検出
感度、精度の高い細菌検出が可能となる効果が得られ
る。According to the second aspect of the present invention, in particular, the filtration of the phage solution is performed from the backwash direction with respect to the filtration of the sample liquid by using a filtration membrane. In addition, the noise component contained in the phage solution can be effectively removed, and an effect that enables detection of bacteria with higher detection sensitivity and accuracy can be obtained.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) C12R 1:19) (C12Q 1/68 C12R 1:19) (72)発明者 野上 尊子 東京都江東区新砂1丁目2番8号 オルガ ノ株式会社内 (72)発明者 明賀 春樹 東京都江東区新砂1丁目2番8号 オルガ ノ株式会社内 (72)発明者 平岡 睦久 神奈川県川崎市川崎区田辺新田1番1号 富士電機株式会社内 (72)発明者 野田 直広 神奈川県川崎市川崎区田辺新田1番1号 富士電機株式会社内 (72)発明者 大戸 時喜雄 神奈川県川崎市川崎区田辺新田1番1号 富士電機株式会社内 (72)発明者 多田 弘 神奈川県川崎市川崎区田辺新田1番1号 富士電機株式会社内 Fターム(参考) 4B029 AA07 BB02 BB13 CC01 FA01 FA09 4B063 QA01 QQ06 QR41 QR66 QR79 QS12 QX02 ──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 Identification FI FI Theme Court II (Reference) C12R 1:19) (C12Q 1/68 C12R 1:19) (72) Inventor Takako Nogami Shinsuna, Koto-ku, Tokyo 1-2-8, Organo Co., Ltd. (72) Inventor Haruki Akiga 1-2-2-8, Shinsuna, Koto-ku, Tokyo Organo Co., Ltd. (72) Inventor Mutsuhisa Hiraoka Tanabe Nitta, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture No. 1-1 Inside Fuji Electric Co., Ltd. (72) Inventor Naohiro Noda 1-1, Tanabe Nitta, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Inside Fuji Electric Co., Ltd. (72) Inventor Tokio Oto Arata Tanabe, Kawasaki-ku, Kawasaki-shi, Kanagawa Fuji Electric Co., Ltd. (72) Inventor Hiroshi Tada 1-1-1, Tanabe-Nitta, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture F-term (reference) 4B029 AA07 BB02 BB13 CC01 FA01 FA09 4B063 QA01 QQ06 QR41 QR66 QR79 QS12 QX02
Claims (8)
テリオファージを宿主細菌に接触させることで該標識核
酸を宿主細菌に注入して細菌を染色し、この染色した状
態を光学的に検出する細菌の検出方法において、測定対
象の細菌を含む試料液をろ過材でろ過することで、該試
料液中の細菌をろ過材で捕捉すると共に、試料液に含ま
れる色素及び/又は蛍光物質を除去し、この後、上記宿
主細菌に特異的でかつ上記核酸標識したバクテリオファ
ージをろ過材上に捕捉した細菌に接触させて上記染色を
行わせ、この染色した細菌を計数することを特徴とする
細菌の検出方法。1. A bacterium which contacts a host bacterium with a bacteriophage labeled with a dye or a fluorescent substance to inject the labeled nucleic acid into the host bacterium and stains the bacterium, and optically detects the stained state. In the detection method, the sample liquid containing the bacteria to be measured is filtered through a filter medium, whereby the bacteria in the sample liquid are captured by the filter medium, and the dye and / or fluorescent substance contained in the sample liquid are removed. Thereafter, the bacteriophage specific to the host bacterium and labeled with the nucleic acid is brought into contact with a bacterium captured on a filter medium to perform the staining, and the number of the stained bacterium is counted. Detection method.
を用い、試料液の通液ろ過により該試料液に含まれる細
菌の捕捉と色素及び/又は蛍光物質の除去とを行なった
後、このろ過膜に対し試料液の通液ろ過方向とは反対方
向に上記核酸を標識したバクテリオファージを含むファ
ージ溶液を通液ろ過してバクテリオファージを細菌に接
触させることで上記染色を行わせることを特徴とする細
菌の検出方法。2. The method according to claim 1, wherein a filtration membrane is used as a filtration material, and bacteria contained in the sample solution and dyes and / or fluorescent substances are removed by filtration of the sample solution. A phage solution containing a bacteriophage labeled with the nucleic acid is filtered through a phage solution containing the bacteriophage labeled in the direction opposite to the filtration direction of the sample solution through the filtration membrane, and the staining is performed by contacting the bacteriophage with the bacteria. Bacteria detection method.
テリオファージの宿主細菌に接触させることで該標識核
酸を宿主細菌に注入して細菌を染色し、この染色した状
態を光学的に検出する細菌の方法において、宿主細菌に
特異的でかつ上記核酸標識したバクテリオファージを含
むファージ溶液を測定対象の細菌を含む試料液に添加し
て該バクテリオファージを細菌に接触させることで上記
染色を行わせ、この溶液をろ過材でろ過して上記染色し
た細菌を捕捉すると共に、該溶液に含まれる色素及び/
又は蛍光物質を除去した後、染色した細菌を計数するこ
とを特徴とする細菌の検出方法。3. A bacterium which contacts a host bacterium of a bacteriophage whose nucleic acid is labeled with a dye or a fluorescent substance, injects the labeled nucleic acid into the host bacterium, stains the bacterium, and optically detects the stained state. In the method, the phage solution containing a bacteriophage specific for the host bacteria and the nucleic acid is added to a sample solution containing the bacteria to be measured, and the bacteriophage is subjected to the staining by contacting the bacteria, The solution is filtered through a filter medium to capture the stained bacteria, and the dye and / or dye contained in the solution is trapped.
Alternatively, a method for detecting bacteria, which comprises counting stained bacteria after removing a fluorescent substance.
膜であることを特徴とする細菌の検出方法。4. The method for detecting bacteria according to claim 1, wherein the filtration material is a filtration membrane.
バクテリオファージの核酸を標識する色素又は蛍光物質
で染色された細菌を捕捉しているろ過材上の染色点を、
光学的検出手段により測定することで細菌数を計数する
ことを特徴とする細菌の検出方法。5. The method according to claim 1, wherein
A staining point on a filter medium capturing bacteria stained with a dye or a fluorescent substance that labels bacteriophage nucleic acids,
A method for detecting bacteria, wherein the number of bacteria is counted by measuring with an optical detection means.
回収液によるろ過材の洗浄あるいは回収液へのろ過材の
浸漬により、該ろ過材に捕捉されている細菌を回収液中
に分離回収し、この回収液中の染色細菌を光学的に検出
して計数することを特徴とする記載の細菌の検出方法。6. The method according to claim 1, wherein
By washing the filter medium with the recovery liquid or immersing the filter medium in the recovery liquid, the bacteria trapped in the filter medium are separated and recovered in the recovery liquid, and the stained bacteria in the recovery liquid are optically detected. The method for detecting bacteria according to the above, characterized by counting.
ろ過膜の逆洗あるいは回収液中へのろ過膜の浸漬によ
り、該ろ過膜に捕捉されている細菌を回収液中に分離回
収し、この回収液中の染色細菌を光学的に検出して計数
することを特徴とする細菌の検出方法。7. The method according to claim 2, wherein the bacteria trapped in the filtration membrane are separated and recovered in the recovery liquid by backwashing the filtration membrane with the recovery liquid or immersing the filtration membrane in the recovery liquid. A method for detecting bacteria, wherein the stained bacteria in the recovered solution are optically detected and counted.
ろ過する試料液量50ml以上に対し、該ろ過材に捕捉
した細菌を分離回収する回収液量が10ml以下である
ことを特徴とする低濃度試料液中の含有細菌の検出方
法。8. The method according to claim 6, wherein the amount of the collected liquid for separating and recovering the bacteria captured by the filter medium is 10 ml or less for the sample liquid amount of 50 ml or more for passing through the filter medium. For detecting bacteria contained in low-concentration sample solutions.
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JP11083590A JP2000270892A (en) | 1999-03-26 | 1999-03-26 | Detection of bacillus |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003135094A (en) * | 2001-10-30 | 2003-05-13 | Iatron Lab Inc | Novel method for analyzing bacteria |
KR100998888B1 (en) | 2008-11-14 | 2010-12-08 | 한국생명공학연구원 | Evaluation apparatus of air filtration system for reducing the transmission of viruses and method thereof |
JP2014135935A (en) * | 2013-01-17 | 2014-07-28 | Azbil Corp | System and method for detecting microorganism |
JP2018535700A (en) * | 2015-11-20 | 2018-12-06 | シナミラ アーゲー | Method and apparatus for detecting bacteria |
-
1999
- 1999-03-26 JP JP11083590A patent/JP2000270892A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003135094A (en) * | 2001-10-30 | 2003-05-13 | Iatron Lab Inc | Novel method for analyzing bacteria |
KR100998888B1 (en) | 2008-11-14 | 2010-12-08 | 한국생명공학연구원 | Evaluation apparatus of air filtration system for reducing the transmission of viruses and method thereof |
JP2014135935A (en) * | 2013-01-17 | 2014-07-28 | Azbil Corp | System and method for detecting microorganism |
JP2018535700A (en) * | 2015-11-20 | 2018-12-06 | シナミラ アーゲー | Method and apparatus for detecting bacteria |
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