JPH09133684A - Method and reagent for detecting specified bacterium in food - Google Patents
Method and reagent for detecting specified bacterium in foodInfo
- Publication number
- JPH09133684A JPH09133684A JP31582695A JP31582695A JPH09133684A JP H09133684 A JPH09133684 A JP H09133684A JP 31582695 A JP31582695 A JP 31582695A JP 31582695 A JP31582695 A JP 31582695A JP H09133684 A JPH09133684 A JP H09133684A
- Authority
- JP
- Japan
- Prior art keywords
- food
- particles
- antibody
- aggregation
- 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
Links
Landscapes
- 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、食品中の特定細菌
の検出方法および検出試薬に関する。TECHNICAL FIELD The present invention relates to a method and a reagent for detecting specific bacteria in foods.
【０００２】[0002]
【従来の技術】食品の安全性を評価するに際して食品検
査は必要不可欠である。またその結果は食品保健行政
上、また自主管理上の措置の根拠となる。ここ数年来の
食中毒の発生件数はやや減少傾向であるにしても、逆に
患者数は増大している。この事は食品の大型化と流通機
構の発展も関与していると考えられる。安全かつ良質な
食品提供は、生産、流通、販売など食品営業者に課せら
れた社会的責務である。2. Description of the Related Art Food inspection is indispensable for evaluating the safety of food. The results are also the basis for measures for food health administration and voluntary management. Although the number of food poisoning cases has been decreasing for some years, the number of patients is increasing. It is considered that this is also related to the enlargement of food and the development of distribution system. Providing safe and high-quality food is a social responsibility imposed on food business operators such as production, distribution, and sales.
【０００３】食品中の微生物検査は大きく分けて２つの
方向がある。その一つは汚染度合を知る目的で総菌数検
査であり、他の一つは、食中毒原因菌すなわち特定細菌
の有無を確定する検査である。特定細菌の検出には厚生
省生活局監修の食品衛生検査指針「微生物編」にも記載
されている通り、食品にはいわゆる食中毒原因菌のみな
らず規制以外の微生物の混在する場合がある。従って、
特定細菌検査には目的菌がある程度選択的に発育する分
離培地による分離培養と、目的菌と疑う集落を更に、生
化学的、血清学的試験をもとに菌種を決定する操作が必
要である。また少量の病原菌汚染の場合には、分離培養
の前に更に液体培地で前培養を行う必要がある。例え
ば、サルモネラを検出する場合、試料をＥＥＭブイヨン
等で前増菌培養後、ＳＢＧ培地等により増菌培養を行
い、続いてＭＬＣＢ寒天培地等で分離培養を行う。この
時点でサルモネラによる汚染が疑わしい集落について、
更にＴＳＩ寒天培地等により確認培養を行い、ＶＰ半流
動培地等によって同定を行い、市販血清で血清型別を確
認する。ここまでの検査工程で５日間以上の長時間を要
する。Microbial tests in foods are roughly divided into two directions. One of them is a total bacterial count test for the purpose of knowing the degree of contamination, and the other is a test for determining the presence or absence of food poisoning causative bacteria, that is, specific bacteria. For the detection of specific bacteria, as described in the Food Sanitation Inspection Guideline "Microbial Edition" supervised by the Ministry of Health and Welfare, the food may contain not only so-called food poisoning bacteria but also microorganisms other than those regulated. Therefore,
For specific bacteria testing, it is necessary to carry out separation culture using a separation medium in which the target bacteria grow selectively to some extent, and operations to determine the bacterial species based on biochemical and serological tests in addition to the colonies suspected to be the target bacteria. is there. When a small amount of pathogenic bacteria is contaminated, it is necessary to further perform preculture in a liquid medium before the separation culture. For example, when Salmonella is detected, the sample is subjected to pre-enrichment culture with EEM broth or the like, followed by enrichment culture with SBG medium or the like, and then separate culture with MLCB agar medium or the like. At this point, about the village suspected to be contaminated by Salmonella,
Further, confirmation culture is performed using TSI agar medium or the like, identification is performed using VP semi-fluid medium or the like, and serotype is confirmed with commercially available serum. The inspection process so far requires a long time of 5 days or more.
【０００４】[0004]
【発明が解決しようとする課題】前記の一般的食品検査
において、特定細菌を検索しようとする場合、分離培養
後、目的とする細菌を疑う集落をさらに生化学的性状や
血清学的性状により同定を行って検査が終了する。この
結果判定までの所要時間は、早くても３〜４日間、ある
いはそれ以上の日数が必要なのが現状である。従って新
鮮度を必要とする食品の場合は、検査結果を待たずに出
荷される場合があり、また検査終了を待つ場合は、食品
を特定条件で保管する必要があり、その設備と多額の保
管料が必要となり、コストアップの原因ともなる。本発
明は、これらの問題点に着目して行ったものであって、
抗原抗体反応により凝集反応を用いて、食品中の特定細
菌検出を迅速且つ簡便に行う事のできる方法とそのため
の試薬を提供する事を目的とする。When searching for a specific bacterium in the above general food test, after isolation and culturing, a colony suspected of the target bacterium is further identified by biochemical properties or serological properties. And the inspection ends. At present, the time required for this result determination is at least 3 to 4 days, or more. Therefore, in the case of food that requires freshness, it may be shipped without waiting for the inspection result, and when waiting for the end of the inspection, it is necessary to store the food under specific conditions. Fees are required, which also increases costs. The present invention was carried out focusing on these problems,
It is an object of the present invention to provide a method capable of detecting specific bacteria in food quickly and easily by using an agglutination reaction by an antigen-antibody reaction, and a reagent therefor.
【０００５】[0005]
【課題を解決するための手段】従って、本発明は、免疫
学的測定による食品微生物検査において、食品あるいは
その処理物と目的細菌と特異的に結合する抗体を感作し
た不溶性粒子を接触させて、抗原抗体反応により生じる
凝集を検出することを特徴とする食品中の特定細菌の検
査方法に、関する。さらに、少なくとも食品あるいはそ
の処理物中の目的細菌と特異的に結合する抗体を感作し
た不溶性粒子からなる食品中の特定細菌の検査試薬、に
も関する。SUMMARY OF THE INVENTION Therefore, according to the present invention, in a food microbiological test by immunological measurement, food or a processed product thereof is brought into contact with insoluble particles sensitized with an antibody that specifically binds to a target bacterium. The present invention relates to a method for inspecting specific bacteria in food, which comprises detecting aggregation caused by an antigen-antibody reaction. Further, it relates to a test reagent for a specific bacterium in a food, which comprises insoluble particles sensitized with an antibody that specifically binds to a target bacterium in the food or a processed product thereof.
【０００６】[0006]
【発明の実施の形態】本発明における試料としては、乳
及び乳製品、肉及び肉製品、魚介類及び魚介類製品、冷
凍食品、日配食品、飲料水、飼料及び飼料製品、菓子
類、香辛料等の食品全般をいう。これらをそのままある
いは抽出等を行った処理物を用いる。また、抗体は、目
的の特定細菌（サルモネラ、病原大腸菌、カンピロバク
ター、腸炎ビブリオ、ブドウ球菌、セレウス、リステリ
ア、ボツリヌス、ウェルシュ、エルシニア、赤痢、コレ
ラからなる食中毒原因菌）の加熱及び酸、アルカリ処理
菌体、または特定細菌の菌体成分精製物、あるいは特定
細菌の菌体抽出精製物等を抗原として哺乳動物に免疫し
て得た抗血清（ポリクローナル抗体）、あるいは前記抗
原をマウスまたはラットに免疫したＢ細胞とミエローマ
細胞を融合させて確立したハイブリドーマを動物の腹腔
内に接種し産生された抗体やハイブリドーマの培養上清
を精製して得たモノクローナル抗体を用いることができ
る。BEST MODE FOR CARRYING OUT THE INVENTION As samples in the present invention, milk and dairy products, meat and meat products, seafood and seafood products, frozen foods, daily foods, drinking water, feed and feed products, confectionery, spices. Etc. refers to all food products. These are used as they are or processed products that have been extracted. Antibodies are heat-, acid-, and alkali-treated bacteria of specific bacteria of interest (salmonella, pathogenic Escherichia coli, Campylobacter, Vibrio parahaemolyticus, staphylococcus, cereus, listeria, botulinum, welsh, yersinia, dysentery, cholera). Antiserum (polyclonal antibody) obtained by immunizing a mammal with the body, a purified bacterial cell component of a specific bacterium, or a purified bacterial cell extract of a specific bacterium as an antigen, or immunizing a mouse or rat with the antigen. An antibody produced by inoculating an intraperitoneal cavity of an animal with a hybridoma established by fusing B cells and myeloma cells or a monoclonal antibody obtained by purifying a culture supernatant of the hybridoma can be used.
【０００７】抗体を感作する不溶性粒子としては、赤血
球、天然高分子、合成高分子、ガラス、磁気粒子等を用
いることができる。好ましくは、ラテックス粒子、磁気
粒子であり、それらの粒径は凝集反応に適用できる範囲
（例えば０．０１〜１０μｍ）であれば特に限定は無
い。免疫反応によって凝集した粒子を捕捉する操作を容
易に行うためには、磁気粒子がより好ましい。これら粒
子は既に多数市販されており、適宜選択し用いることが
できる。As the insoluble particles for sensitizing the antibody, erythrocytes, natural polymers, synthetic polymers, glass, magnetic particles and the like can be used. Latex particles and magnetic particles are preferable, and the particle size thereof is not particularly limited as long as it can be applied to the aggregation reaction (for example, 0.01 to 10 μm). Magnetic particles are more preferable for facilitating the operation of capturing particles aggregated by an immune reaction. Many of these particles are already on the market and can be appropriately selected and used.
【０００８】前記で得た抗体を不溶性粒子に感作する方
法は公知であり、例えば疎水結合を利用した物理吸着
法、あるいは活性化させたトシル基、カルボキシル基、
アミノ基、チオール基等で共有結合させる化学結合法を
用いることができる。抗体を感作した粒子は、例えばリ
ン酸緩衝液等の適当な溶媒中で保存できる。A method for sensitizing the above-obtained antibody to insoluble particles is known, for example, a physical adsorption method utilizing a hydrophobic bond, or an activated tosyl group, carboxyl group,
A chemical bonding method of covalently bonding with an amino group, a thiol group, or the like can be used. The antibody-sensitized particles can be stored in a suitable solvent such as a phosphate buffer.
【０００９】食品中の病原性細菌の一般的な検査手順
は、試料を前増菌培養、増菌培養、分離培養の手順を経
た後、更に確認培養後判定する、という行程を経る。対
して、本発明の操作法としては、試料を直接凝集反応系
に導くことを基本とするものである。例えば精肉中のサ
ルモネラ菌を検出する場合、鞭毛抗原をウサギ等で免疫
して得た抗血清を磁気粒子と適当な緩衝液（例えば、ｐ
Ｈ９．５のホウ酸緩衝液等）中で混合し抗体を磁気粒子
に感作し、磁石で粒子を集め、適当な緩衝液（例えば、
牛血清アルブミン０．１％を含むｐＨ７．４のリン酸緩
衝液等）中に懸濁して、抗体感作磁気粒子懸濁液とす
る。精肉２５〜５０ｇ程度を細かく切断して、生理食塩
水やリン酸緩衝液等による洗い出しを行った後の濾過
液、またはブイヨン等を加えて乳化させて濾過した液、
あるいは必要に応じて４〜８時間、３７℃で培養した後
濾過した液を試料液とする。この試料液の一定量（０．
１〜１０ｍｌ）を採取（例えば、試験管や反応ウェル）
に加え、そこに前記抗体感作磁気粒子懸濁液を加えて室
温で５〜６０分間反応させる。次に、磁石を用い磁気粒
子を一定部位（例えば、反応容器の底部等）に集め、上
清を吸引除去する。この時抗体磁気粒子には目的の細菌
のみ吸着し、他の夾雑細菌や食品由来の浮遊物が取り除
かれる。集めた粒子を適当量（例えば、５０ｕｌ〜１ｍ
ｌ程度）の緩衝液に再懸濁し、室温で緩やかに回転させ
ながら１〜１０分間反応させ、凝集の有無を判定する。[0009] A general inspection procedure for pathogenic bacteria in foods is such that a sample is subjected to a pre-enrichment culture, an enrichment culture, a separation culture, and then a confirmation culture is performed for determination. On the other hand, the operation method of the present invention is based on direct introduction of the sample into the agglutination reaction system. For example, when detecting Salmonella in meat, the antiserum obtained by immunizing a rabbit or the like with flagella antigen is magnetic particles and an appropriate buffer solution (for example, p
The antibody is sensitized to the magnetic particles by mixing them in H9.5 borate buffer, etc., and the particles are collected with a magnet.
An antibody-sensitized magnetic particle suspension is prepared by suspending the antibody-sensitized magnetic particles in 0.1% bovine serum albumin (pH 7.4 phosphate buffer, etc.). About 25 to 50 g of meat is finely cut and filtered after washing with physiological saline or phosphate buffer, or a liquid obtained by emulsifying with broth etc. and filtering,
Alternatively, if necessary, the solution obtained by culturing at 37 ° C. for 4 to 8 hours and then filtering is used as a sample solution. A fixed amount of this sample solution (0.
1 to 10 ml) (for example, test tube or reaction well)
In addition, the antibody-sensitized magnetic particle suspension is added thereto and reacted at room temperature for 5 to 60 minutes. Next, magnetic particles are collected at a certain site (for example, the bottom of the reaction container) using a magnet, and the supernatant is removed by suction. At this time, only the target bacteria are adsorbed on the antibody magnetic particles, and other contaminant bacteria and food-derived suspended matter are removed. Collect a suitable amount of particles (eg 50ul-1m)
1) buffer solution, and allowed to react for 1 to 10 minutes while gently rotating at room temperature to determine the presence or absence of aggregation.
【００１０】本発明において、食品中の病原微生物は一
般細菌、例えば大腸菌群の汚染度に比較して微量である
か、または損傷を受けている場合があるため、必要に応
じて増菌培養を行い菌数を増やしたうえで凝集反応を行
うことができる（例えば、サルモネラの検査において
は、必要に応じて前増菌培養を行う）。即ち、汚染度が
極微量であっても、これらを必ず検出する必要がある。
そのため、検査結果の信頼性を高めるために、検体試料
を検出目的の細菌に適した増菌培地であらかじめ培養
し、菌数を増やしてから抗体粒子による凝集反応を行う
ことが好ましい。この増菌培養は、従来から用いられて
いる培地種と培養条件をそのまま用いる事が出来る。い
うまでもなく培地種と培養条件は目的細菌によって異な
り、適宜選択することができる。In the present invention, the pathogenic microorganisms in the food may be present in a trace amount or damaged as compared with the degree of contamination of general bacteria such as coliforms. The agglutination reaction can be performed after increasing the number of bacteria (for example, in the examination of Salmonella, pre-enrichment culture is performed as necessary). That is, even if the degree of contamination is extremely small, it is necessary to detect them.
Therefore, in order to increase the reliability of the test result, it is preferable to pre-incubate the specimen sample in an enrichment medium suitable for the bacteria to be detected, increase the number of bacteria, and then perform the agglutination reaction with the antibody particles. For this enrichment culture, the conventionally used medium species and culture conditions can be used as they are. Needless to say, the medium type and culture conditions differ depending on the target bacteria and can be appropriately selected.
【００１１】前記の菌培養で用いられるサルモネラの前
増菌培地としては、ＥＥＭブイヨン、乳糖ブロス、緩衝
ペプトン水等が挙げられる。Examples of the Salmonella pre-enrichment medium used in the above-mentioned bacterial culture include EEM broth, lactose broth, buffered peptone water and the like.
【００１２】また、増菌培地としては、目的細菌毎に公
知のものを使用することができる。例えば、サルモネラ
用としてはセレナイトブリリアントグリーン培地、ラパ
ポートバジリアディス培地、セレナイトシスチン培地等
が挙げられる。また、カンピロバクター用としてはＭａ
ｒｔｉｎカンピロバクター増菌ブイヨン、Ｐｒｅｓｔｏ
ｎブイヨン、Ｄｏｙｌｅ−Ｒｏｍａｎ培地等が挙げられ
る。また、腸炎ビブリオ用としては、アルカリ性ペプト
ン水、食塩ポリミキシンブイヨン等が挙げられる。ま
た、ブドウ球菌用としては食塩加トリプトケース−ソイ
ブイヨン等が挙げられる。また、セレウス用としてはポ
リミキシン加トリプトケースソイブイヨン等が挙げられ
る。また、リステリア用としてはリステリア増菌培地等
が挙げられる。また、ボツリヌス用としてはクックトミ
ート培地等が挙げられる。また、エルシニア用としては
ソルビット、胆汁酸塩加リン酸緩衝生理食塩水等が挙げ
られる。赤痢用としてはグラムネガティブブイヨン、Ｃ
ＰＰＧブイヨン等が挙げられる。また、コレラ用として
はアルカリ性ペプトン水等が挙げられる。これら以外に
も目的細菌毎に増菌培地を適宜選択しうる。As the enrichment medium, known ones can be used for each target bacterium. For example, for Salmonella, a selenite brilliant green medium, a rapaport vasilidis medium, a selenite cystine medium and the like can be mentioned. Ma for Campylobacter
rtin Campylobacter enriched broth, Presto
n broth, Doyle-Roman medium and the like. For Vibrio parahaemolyticus, alkaline peptone water, salt polymyxin broth and the like can be mentioned. For Staphylococcus, tryptocase-soy broth supplemented with sodium chloride and the like can be mentioned. For cereus, polymyxin-containing tryptocase soy broth and the like can be mentioned. For Listeria, Listeria enriched medium and the like can be mentioned. For botulinum, Cook's meat medium and the like can be mentioned. For Yersinia, sorbit, bile salt-added phosphate buffered saline, etc. may be mentioned. For dysentery Gram negative broth, C
Examples thereof include PPG broth. For cholera, alkaline peptone water and the like can be mentioned. Besides these, an enrichment medium can be appropriately selected for each target bacterium.
【００１３】こうして得た試料液と前記抗体感作粒子懸
濁液を接触混合させ、抗原抗体反応を行う。それによっ
て発現した凝集を目視あるいは光学的手段により検出す
る。このとき、より凝集を鮮明にするために、前述の通
り、抗原抗体反応によって凝集した粒子を分別すること
が好ましい。不溶性粒子として磁気粒子を用いた場合に
は、磁石によって粒子を捕捉（集菌）して、これを適当
な溶液（例えば、リン酸緩衝液、グッド緩衝液、グリシ
ン緩衝液、トリス緩衝液等の緩衝液）に再懸濁して凝集
を検出することができる。また、不溶性粒子としてポリ
スチレン等のラテックスを用いた場合には、濾別して凝
集した粒子を集め、これを適当な溶液に再懸濁して凝集
を検出することもできる。The sample solution thus obtained and the antibody-sensitized particle suspension are contact-mixed to carry out an antigen-antibody reaction. The aggregation thus developed is detected visually or by optical means. At this time, in order to make the aggregation clearer, it is preferable to separate the particles aggregated by the antigen-antibody reaction as described above. When magnetic particles are used as the insoluble particles, the particles are captured (collected by a magnet) by a magnet, and the particles are collected in an appropriate solution (for example, phosphate buffer, Good's buffer, glycine buffer, Tris buffer, etc.). It can be resuspended in a buffer) to detect aggregation. When latex such as polystyrene is used as the insoluble particles, the aggregated particles can be collected by filtration and resuspended in an appropriate solution to detect aggregation.
【００１４】更に、前記で得た目的の菌と特異的に結合
する抗体を感作した不溶性粒子を懸濁させた緩衝液を、
特定細菌の検出用試薬とすることができる。このとき
の、抗体の感作量、懸濁液中に含まれる不溶性粒子の含
有量は特には限定されず、目的の菌、抗体の力価、不溶
性粒子の種類によって適宜選択することが可能である。
更に、緩衝液には防腐剤としてアジ化ナトリウム、トッ
プサイド等を含有することができる。また、分散剤ある
いは安定化剤として界面活性剤を含有させることもでき
る。また、前記の培地と組み合わせて、特定細菌の検出
用キットとしても構成することが可能である。Further, a buffer solution in which insoluble particles sensitized with the antibody specifically binding to the target bacterium obtained above are suspended,
It can be used as a reagent for detecting specific bacteria. At this time, the sensitizing amount of the antibody, the content of the insoluble particles contained in the suspension is not particularly limited, it can be appropriately selected depending on the target bacteria, the titer of the antibody, the type of the insoluble particles. is there.
Further, the buffer solution may contain sodium azide, topside, etc. as a preservative. Further, a surfactant can be contained as a dispersant or a stabilizer. Further, it can be configured as a kit for detecting a specific bacterium by combining with the above-mentioned medium.
【００１５】以下、実施例により本発明を具体的に説明
するが、これらは本発明の範囲を限定するものではな
い。 実施例：市販鶏肉中のサルモネラ菌の検出 （Ａ）抗体感作粒子の調製 自家製サルモネラ鞭毛抗原をｐＨ７．４の１／１５Ｍリ
ン酸緩衝液に懸濁した液を、ウサギに皮下免疫した。１
週間後再度同様に免疫し、計４回の免疫操作を行った。
免疫開始５週目に採血して血清分離を行い抗血清を得
た。上記抗血清を磁気粒子（外径２．８μｍ、ダイナル
社）１０7〜１０8個／ｍｌを含むｐＨ９．５のホウ酸緩
衝液と等量混合し、３７℃で２４時間回転混和した。磁
石で抗体感作磁気粒子を集め、上清を吸引除去し、牛血
清アルブミン０．１％を含むｐＨ７．４のリン酸緩衝液
で４回洗浄し、更に前記リン酸緩衝液を加えて４℃で一
晩放置した。磁気粒子を磁石で集め、上清を吸引除去
し、０．０２％のアジ化ナトリウム、０．１％の牛血清
アルブミンを含むｐＨ７．４のリン酸緩衝液に懸濁し、
抗体感作磁気粒子懸濁液とした。Hereinafter, the present invention will be described in detail with reference to Examples, but these do not limit the scope of the present invention. Example: Detection of Salmonella bacterium in commercial chicken (A) Preparation of antibody-sensitized particles A rabbit was subcutaneously immunized with a solution prepared by suspending homemade Salmonella flagella antigen in a 1/15 M phosphate buffer of pH 7.4. 1
After a week, the same immunization was performed again, and a total of four immunization operations were performed.
Five weeks after the start of immunization, blood was collected and serum was separated to obtain antiserum. The above antiserum was mixed in an equal amount with a borate buffer solution of pH 9.5 containing 10 7 to 10 8 magnetic particles (outer diameter 2.8 μm, Dynal Co., Ltd.) / Ml, and rotatively mixed at 37 ° C. for 24 hours. The antibody-sensitized magnetic particles were collected with a magnet, the supernatant was removed by suction, and the particles were washed 4 times with a phosphate buffer solution containing 0.1% bovine serum albumin at pH 7.4. Left overnight at ° C. The magnetic particles were collected with a magnet, the supernatant was removed by suction, and the suspension was suspended in a phosphate buffer of pH 7.4 containing 0.02% sodium azide and 0.1% bovine serum albumin.
An antibody-sensitized magnetic particle suspension was prepared.
【００１６】（Ｂ）試料液の調製 市販鶏肉１００検体について、それぞれの２５ｇを、滅
菌した１／１５Ｍリン酸緩衝液５０ｍｌで洗い出し、そ
の液を乾燥ブイヨン２００ｍｌに加え、３７゜Ｃ、６時
間培養した培養液（前増菌培養）１ｍｌをラパポートバ
ジリアディスサルモネラ増菌ブイヨン（メルク社）１０
ｍｌに加え、４２゜Ｃで２４時間培養（選択増菌培養）
し、この培養液を凝集反応の試料とした。凝集反応は、
反応ウェルに抗体感作磁気粒子懸濁液３０μｌをとり、
上記試料１ｍｌを加え、室温で１０分間、撹袢しながら
反応させた。続いて磁石で磁気粒子を集め、上清を吸引
除去する。０．０５％ツイーン２０を含むｐＨ７．４の
０．０１Ｍリン酸緩衝液１ｍｌを加え、磁気粒子を再懸
濁後、再び磁気粒子を集め、上清を吸引除去後、０．０
５％ツイーン２０を含むｐＨ７．４の０．０１Ｍリン酸
緩衝液１５０μｌを加えて緩やかに撹袢し、凝集の有無
を肉眼で判定した。(B) Preparation of sample solution For 100 samples of commercially available chicken meat, 25 g of each was washed out with 50 ml of sterilized 1 / 15M phosphate buffer, and the solution was added to 200 ml of dry broth and incubated at 37 ° C for 6 hours. 1 ml of the prepared culture solution (pre-enrichment culture) was added to Rapaport Vagilia Dissalmonella Enrichment Broth (Merck) 10
In addition to ml, culture at 42 ° C for 24 hours (selective enrichment culture)
Then, this culture solution was used as a sample for the agglutination reaction. The agglutination reaction is
Take 30 μl of antibody-sensitized magnetic particle suspension in a reaction well,
1 ml of the above sample was added, and the mixture was reacted at room temperature for 10 minutes with stirring. Subsequently, the magnetic particles are collected with a magnet, and the supernatant is removed by suction. 1 ml of 0.01 M phosphate buffer solution containing 0.05% Tween 20 at pH 7.4 was added to resuspend magnetic particles, and then the magnetic particles were collected again, and the supernatant was removed by suction.
150 μl of 0.01 M phosphate buffer of pH 7.4 containing 5% Tween 20 was added and gently stirred, and the presence or absence of aggregation was visually determined.
【００１７】（Ｃ）培養法１ 上記操作に用いた選択増菌培養液を、本発明法の成績が
サルモネラによるものかを確認する方法としてマンニッ
ト−リシン−クリスタルバイオレット−ブリリアントグ
リーン（ＭＬＣＢ培地）（日水製薬）で３７゜Ｃ、２４
時間分離培養を行い、黒色コロニー（即ち、サルモネラ
を疑われる集落）をＴＳＩ寒天培地（日水製薬）、ＳＩ
Ｍ確認培地（日水製薬）、リジン鉄寒天培地（ＢＢＬ
社）の３種類の確認培地に接種して、３７℃で２４時間
培養後、市販簡易キット（商品名：Ａｐｉ２０Ｅ、日本
ビオメリュー社）で同定した。(C) Culturing Method 1 Mannite-lysine-crystal violet-brilliant green (MLCB medium) was used as a method for confirming whether the selective enrichment culture solution used in the above operation was due to Salmonella. (Nissui Pharmaceutical) at 37 ° C, 24
Time-separated culture is performed, and black colonies (ie, colonies suspected of Salmonella) are subjected to TSI agar (Nissui Pharmaceutical), SI
M confirmation medium (Nissui Pharmaceutical), lysine iron agar medium (BBL
Incubating for 24 hours at 37 ° C., and then identified with a commercially available simple kit (trade name: Api20E, Japan BioMerieux Co., Ltd.).
【００１８】（Ｄ）比較例：培養法２［従来法：食品衛
生検査指針（厚生省生活局監修）による］ 市販鶏肉２５ｇを滅菌した１／１５Ｍリン酸緩衝液５０
ｍｌで洗い出し、その液をＥＥＭブイヨン（日水製薬）
２００ｍｌに加え、３５゜Ｃ、１８時間培養した培養液
（前増菌培養）１ｍｌをＳＢＧ培地（日水製薬）１０ｍ
ｌに加え、４３゜Ｃ、１８時間培養（選択増菌培養）
し、その培養液をＴＳＩ寒天培地（日水製薬）、ＳＩＭ
確認培地（日水製薬）、リジン鉄寒天培地（ＢＢＬ社）
の３種類の確認培地に接種して、３７℃で２４時間培養
後、市販簡易キット（商品名：Ａｐｉ２０Ｅ、日本ビオ
メリュー社）で同定した。(D) Comparative Example: Culture Method 2 [Conventional Method: According to Food Sanitation Inspection Guidelines (Supervised by the Ministry of Health and Welfare Bureau)] 1/15 M phosphate buffer 50 sterilized with 25 g of commercially available chicken
Rinse out with ml and use the solution as an EEM broth (Nissui Pharmaceutical)
In addition to 200 ml, 1 ml of the culture solution (pre-enrichment culture) that was cultured at 35 ° C for 18 hours was added with 10 m of SBG medium (Nissui Pharmaceutical).
In addition to 1, culture at 43 ° C for 18 hours (selective enrichment culture)
Then, the culture solution is added to TSI agar medium (Nissui Pharmaceutical), SIM
Confirmation medium (Nissui Pharmaceutical), lysine iron agar medium (BBL)
Was inoculated into the three types of confirmation mediums described above, cultured at 37 ° C. for 24 hours, and then identified with a commercially available simple kit (trade name: Api20E, Japan BioMerieux).
【００１９】（Ｅ）結果 同一試料を検体として、本発明法と、培養法を用い、鶏
肉１００検体からサルモネラを検出した結果を下記に示
した。(E) Results The results of detecting Salmonella from 100 chicken samples using the method of the present invention and the culture method using the same sample as the sample are shown below.
【００２０】[0020]
【表１】 [Table 1]
【００２１】表１の如く、陽性一致率が４４例、陰性一
致率が５５例、培養法陽性、本発明法陰性が一検体あっ
たが、その逆は一例もなかった。また、不一致の一検体
について、生化学的、また血清学的試験の結果、サルモ
ネラと同定された。従って、本発明法においては一例の
みが正確に検出できなかったものの、培養法１と本発明
法との一致率は９９％と満足のいく結果であった。更
に、感度は９７．８％、特異性は１００％を示した。以
上の結果から、本発明法は、簡単な前増菌培養と増菌培
養を行うだけで凝集系の検出に導くことができ、本来必
要な分離培養及び確認培養等を行わず培養法に匹敵する
精度を得ることができる。また、本発明では、前増菌培
養のみを行い、これを検出系に導いても充分にサルモネ
ラを検出することができた。更に、菌数が充分に多いも
のは、洗い出し液を直接検出系に導いてもサルモネラを
検出することができる検体も存在した。As shown in Table 1, there were 44 positive concordance rates, 55 negative concordance rates, one culture method positive and one negative method according to the present invention, but not the opposite. In addition, one inconsistent sample was identified as Salmonella as a result of biochemical and serological tests. Therefore, in the method of the present invention, although only one example could not be detected accurately, the concordance rate between the culture method 1 and the method of the present invention was 99%, which was a satisfactory result. Furthermore, the sensitivity was 97.8% and the specificity was 100%. From the above results, the method of the present invention can lead to the detection of the agglutination system by simply performing the pre-enrichment culture and the enrichment culture, and is comparable to the culture method without performing the necessary isolation culture and confirmation culture. It is possible to obtain accuracy. Further, in the present invention, salmonella could be sufficiently detected even if only pre-enrichment culture was carried out and this was introduced into the detection system. Furthermore, in the case of a sufficiently large number of bacteria, there was also a sample capable of detecting Salmonella even if the wash-out solution was directly introduced into the detection system.
【００２２】本発明法が、サルモネラを迅速・簡便に検
出することができる方法であることを更に確認するため
に、本発明法と比較例（従来法）との鶏肉１００検体お
けるサルモネラ陽性数の比較を表２に示す。In order to further confirm that the method of the present invention is a method capable of detecting Salmonella rapidly and simply, the number of Salmonella positive counts in 100 chicken samples of the method of the present invention and a comparative example (conventional method) The comparison is shown in Table 2.
【００２３】[0023]
【表２】 [Table 2]
【００２４】表２の如く、本発明法における鶏肉１００
検体中のサルモネラ陽性数は４４検体であり、対して比
較例によるサルモネラ陽性数は４３検体であった。従っ
て、本発明は、充分迅速簡便に市販鶏肉のサルモネラを
検出することができることが確認された。As shown in Table 2, 100 chicken according to the method of the present invention
The number of Salmonella positives in the sample was 44, whereas the number of Salmonella positives in the comparative example was 43. Therefore, it was confirmed that the present invention can detect Salmonella in commercially available chickens sufficiently quickly and easily.
【００２５】市販鶏肉以外に、液卵中のサルモネラにつ
いても同様の試験を行ったところ、正確にサルモネラを
検出することができた。また、サルモネラ以外の食中毒
原因菌も、抗体を変更（必要に応じて目的の菌を公知の
手段によって増菌培養する行程を付加）するだけで、目
的とする菌を容易に検出することができる。In addition to commercially available chicken, the same test was conducted on Salmonella in liquid egg, and Salmonella could be detected accurately. In addition, as for bacteria causing food poisoning other than Salmonella, the target bacterium can be easily detected only by changing the antibody (adding a process for culturing the bacterium of the target bacterium by known means if necessary). .
【００２６】[0026]
【発明の効果】以上説明したように、食品の洗い出し液
や簡単な前増菌等を行ったものを試料として、抗体感作
不溶性粒子（例えば磁気粒子やラテックス）を用いた凝
集法で直接特定細菌を検出する本発明法は、従来行われ
ていた多数の培養ステップを経た方法に匹敵する感度を
有する。また、充分満足のいく一致率が得られ、且つ特
異性も好結果を得ている。更に、細菌汚染の有無の結果
が得られるまでの迅速性に関しても大幅な短縮が可能と
なり、試験を開始してから、たとえ前増菌培養、選択増
菌培養を併用しても翌日には判定結果が得られる。EFFECTS OF THE INVENTION As described above, a product obtained by washing out a food or a simple pre-enrichment is used as a sample and directly identified by an agglutination method using antibody-sensitized insoluble particles (for example, magnetic particles or latex). The method of the present invention for detecting bacteria has a sensitivity comparable to the method that has been conventionally performed through a large number of culture steps. In addition, a sufficiently satisfactory matching rate was obtained, and the specificity was also good. Furthermore, the rapidity of obtaining the results of the presence or absence of bacterial contamination can be significantly shortened, and even if pre-enrichment culture and selective enrichment culture are used together, it will be judged on the next day after the start of the test. The result is obtained.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/543 ５４１ Ｇ０１Ｎ 33/543 ５４１Ａ 33/553 33/553 ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Office reference number FI Technical display location G01N 33/543 541 G01N 33/543 541A 33/553 33/553
Claims (6)
いて、食品あるいはその処理物と目的細菌と特異的に結
合する抗体を感作した不溶性粒子を接触させて、抗原抗
体反応により生じる凝集を検出することを特徴とする食
品中の特定細菌の検査方法。1. In a food microbiological test by immunological measurement, food or a processed product thereof is brought into contact with insoluble particles sensitized with an antibody that specifically binds to a target bacterium, and aggregation caused by an antigen-antibody reaction is detected. A method for inspecting a specific bacterium in food, which is characterized by the following.
検査方法。2. The inspection method according to claim 1, wherein the insoluble particles are magnetic particles.
て磁気粒子以外の夾雑物を除去した後、液体中に該磁気
粒子を再懸濁して凝集を検出する請求項２の検出方法。3. The detection method according to claim 2, wherein after the antigen-antibody reaction, the magnetic particles are captured by a magnet to remove impurities other than the magnetic particles, and then the magnetic particles are resuspended in a liquid to detect aggregation.
ンピロバクター、腸炎ビブリオ、ブドウ球菌、セレウ
ス、リステリア、ボツリヌス、ウェルシュ、エルシニ
ア、赤痢、コレラからなる食中毒原因菌である請求項１
の検出方法。4. The food-borne bacterium comprising Salmonella, pathogenic Escherichia coli, Campylobacter, Vibrio parahaemolyticus, Staphylococcus, Cereus, Listeria, Botulinum, Welsh, Yersinia, dysentery, and cholera.
Detection method.
あらかじめ培養し、その培養液を試料とする請求項１の
検査方法。5. The inspection method according to claim 1, wherein the food or the processed product thereof is previously cultured in an appropriate medium and the culture solution is used as a sample.
目的細菌と特異的に結合する抗体を感作した不溶性粒子
からなる食品中の特定細菌の検査試薬。6. A reagent for testing a specific bacterium in a food, which comprises insoluble particles sensitized with an antibody that specifically binds to a target bacterium in the food or a processed product thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31582695A JPH09133684A (en) | 1995-11-10 | 1995-11-10 | Method and reagent for detecting specified bacterium in food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31582695A JPH09133684A (en) | 1995-11-10 | 1995-11-10 | Method and reagent for detecting specified bacterium in food |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH09133684A true JPH09133684A (en) | 1997-05-20 |
Family
ID=18070036
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP31582695A Pending JPH09133684A (en) | 1995-11-10 | 1995-11-10 | Method and reagent for detecting specified bacterium in food |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH09133684A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010501075A (en) * | 2006-08-14 | 2010-01-14 | バイオコントロール システムズ，インコーポレイティド | How to detect pathogens |
| JP2014528072A (en) * | 2011-09-30 | 2014-10-23 | イースト チャイナ ユニバーシティ オブ サイエンス アンド テクノロジー | Method for detecting plasticizers |
| JP2019049455A (en) * | 2017-09-08 | 2019-03-28 | 東芝テック株式会社 | Sample preparation device and sample preparation method |
-
1995
- 1995-11-10 JP JP31582695A patent/JPH09133684A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010501075A (en) * | 2006-08-14 | 2010-01-14 | バイオコントロール システムズ，インコーポレイティド | How to detect pathogens |
| JP2014528072A (en) * | 2011-09-30 | 2014-10-23 | イースト チャイナ ユニバーシティ オブ サイエンス アンド テクノロジー | Method for detecting plasticizers |
| JP2019049455A (en) * | 2017-09-08 | 2019-03-28 | 東芝テック株式会社 | Sample preparation device and sample preparation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tortorello et al. | Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157: H7 in beef | |
| CN107202884B (en) | Based on immunomagnetic beads and MnO2Nanoparticle detects vibrio parahemolyticus kit | |
| Che et al. | Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode | |
| JPH05264553A (en) | Antigen prepared for detecting helicobacter pyroly | |
| Vermunt et al. | Isolation of salmonellas by immunomagnetic separation | |
| KR101753580B1 (en) | Method for Brucella canis antigen | |
| CN107045061B (en) | Based on Magnetic Isolation and quantum dot-labeled vibrio parahemolyticus detection kit | |
| JPH0782021B2 (en) | Non-immunochemical binding of lipopolysaccharide and its sandwich analysis method | |
| DK174032B1 (en) | Kit as well as immunometric dosing method that can be applied to whole cells | |
| Xu et al. | Rapid detection of Campylobacter jejuni using fluorescent microspheres as label for immunochromatographic strip test | |
| US20150241424A1 (en) | Multi-analyte assay | |
| Zhai et al. | Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence | |
| JP3773633B2 (en) | Analysis method and reagent for E. coli O157 | |
| Jung et al. | Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes | |
| KR20200070792A (en) | Composition and method for simultaneous detection of foodborne pathogens using immunomagnetic particle | |
| US20200355682A1 (en) | Apparatus and method for detecting microbial contamination | |
| JPH09133684A (en) | Method and reagent for detecting specified bacterium in food | |
| US20030059839A1 (en) | Method for detecting pathogens using immunoassays | |
| JP6387063B2 (en) | Method for detecting specific substances in milk | |
| JPS60259966A (en) | Method of rapidly detecting microbe and fungi infection | |
| Khan et al. | Cultural and immunological methods for the detection of Campylobacter jejuni: A review | |
| JP4409273B2 (en) | Apparatus and method for rapid detection of microorganisms | |
| JPS62501726A (en) | Immunoassay system for salmonella detection | |
| JPWO2002065131A1 (en) | Methods and reagents for detecting Vibrio bacteria and antibodies used therefor | |
| RU2189253C1 (en) | Diagnosticum for identification of escherichia coli 0157:h7 and method of its preparing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A02 | Decision of refusal |
Effective date: 20040420 Free format text: JAPANESE INTERMEDIATE CODE: A02 |