JPH0354463A - Apparatus for detecting dna segment or the like - Google Patents
Apparatus for detecting dna segment or the likeInfo
- Publication number
- JPH0354463A JPH0354463A JP7733489A JP7733489A JPH0354463A JP H0354463 A JPH0354463 A JP H0354463A JP 7733489 A JP7733489 A JP 7733489A JP 7733489 A JP7733489 A JP 7733489A JP H0354463 A JPH0354463 A JP H0354463A
- Authority
- JP
- Japan
- Prior art keywords
- rotor
- reagent solution
- filter
- reaction
- dna
- 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
- 238000006243 chemical reaction Methods 0.000 claims abstract description 66
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 239000012634 fragment Substances 0.000 claims description 36
- 230000005855 radiation Effects 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 abstract description 16
- 238000005406 washing Methods 0.000 abstract description 9
- 239000003298 DNA probe Substances 0.000 abstract description 6
- 108020003215 DNA Probes Proteins 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000000295 complement effect Effects 0.000 abstract description 2
- 238000004140 cleaning Methods 0.000 description 11
- 238000009396 hybridization Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 238000004020 luminiscence type Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000007599 discharging Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010396 two-hybrid screening Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は，ＤＮＡ診断等に用いられるＤＮＡ断片などを
，効果的に検出するための検出システムに関する．
〔従来技術］
近年，分子遺伝化学的手法の発展に伴って，種々の遺伝
病の病因や発症機構が，ＤＮＡ診断で明らかにされてい
る。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a detection system for effectively detecting DNA fragments used in DNA diagnosis and the like. [Prior Art] In recent years, with the development of molecular genetic chemistry techniques, the etiology and onset mechanisms of various genetic diseases have been clarified through DNA diagnosis.
このＤＮＡ診断は．従来，次のように行われている．即
ち．まず被検体であるヒト，動物等における末梢血，胎
児絨毛などを採取し，そのＤＮＡを制限酵素により切断
する．そして アガロースゲル電気泳動法により．切断
されたＤＮＡ＠片を分子量の順に分画する．次いで，ゲ
ル上の３ｉ Ｄ ＮＡ断片を変性後，サザンプロット法
でニトロセルロースやナイロン製の担体フィルター（濾
紙）に転写（トランスファー）する．
一方．目的とする遺伝子に対応するＤＮＡ （又はＲＮ
Ａ）を標識したＤＮＡブロープ（反応試薬液）を作威し
ておく．そして．上記担体フィルター上において，相補
的な塩基配列をもつ前記ＤＮＡ断片と上記ＤＮＡプロー
ブとのハイブリッドを形成さセる．然る後，上記ＤＮＡ
ブローブに相同なＤＮＡ断片を検出する．この検出は．
上記標識としての蛍光色素，酵素，放射性同位元素など
の呈色光，β線等を検出することにより行なう．（例え
ば，「実験医学Ｊ ｖｏｊ２５， Ｎ（１１ １．
ｌ ９８７．特開昭６１−４７５６４）。This DNA diagnosis... Conventionally, this is done as follows. That is. First, peripheral blood, fetal villi, etc. from a human or animal subject are collected, and the DNA is cut with restriction enzymes. and by agarose gel electrophoresis. Fractionate the cut DNA@pieces in order of molecular weight. Next, the 3i DNA fragment on the gel is denatured and transferred to a carrier filter (filter paper) made of nitrocellulose or nylon using the Southern blot method. on the other hand. DNA (or RN
Prepare a DNA probe (reaction reagent solution) labeled with A). and. On the carrier filter, a hybrid is formed between the DNA fragment having a complementary base sequence and the DNA probe. After that, the above DNA
Detect DNA fragments homologous to the probe. This detection is.
This is done by detecting colored light, beta rays, etc. from fluorescent dyes, enzymes, radioactive isotopes, etc. as the above-mentioned labels. (For example, “Experimental Medicine J voj25, N(11 1.
l 987. Japanese Patent Publication No. 61-47564).
しかして．本発明は，前記ハイブリッド形威後において
前記ＤＮＡプロープと相同なＤＮＡ断片を検出する際の
検出システムに関する．ところで，従来，ＤＮＡ診断に
おける標識としては，前記のごとく，蛍光色素， Ｒｌ
（放射性同位元素），酵素の呈色光などが使われてい
る．また．上記ハイブリッドの形成に当たっては，前記
担体フィルターを，袋状のパッケージ内において．前記
標識したＤＮＡを含む反応試薬液と接触させる．また，
ハイブリッド形威後は，洗浄を行い，次いで．蛍光色素
，Ｒｌはそのシグナルを，また酵素の場合は発色・発光
物質と反応させることにより，発生したシグナルを検出
する．
（解決しようとするＩ題〕
ところで．従来の検出方法では，上記蛍光色素とＤＮＡ
断片との接触反応時間が短いと発光強度か弱くて十分な
検出ができない。一方．接触反応時間が長すぎると発光
強度が強《なりすぎ，ノイズも多くなり．正確な検出が
できなかった。それ故，従来は，適当と思われる接触反
応時間で，担体フィルターを蛍光色素溶液から引き上げ
て，検出していた。However. The present invention relates to a detection system for detecting a DNA fragment homologous to the DNA probe after the hybridization. By the way, conventionally, as a label in DNA diagnosis, as mentioned above, fluorescent dye, Rl
(radioactive isotopes), colored light from enzymes, etc. are used. Also. In forming the above hybrid, the carrier filter is placed in a bag-like package. Contact with a reaction reagent solution containing the labeled DNA. Also,
After cleaning the hybrid model, wash it and then. The signal generated by the fluorescent dye, Rl, is detected by reacting with the signal, and in the case of the enzyme, by reacting with a coloring/luminescent substance. (Problem I to try to solve) By the way, in conventional detection methods, the above fluorescent dye and DNA
If the contact reaction time with the fragment is short, the luminescence intensity will be low and sufficient detection will not be possible. on the other hand. If the contact reaction time is too long, the luminescence intensity will become too strong and there will be a lot of noise. Accurate detection was not possible. Therefore, in the past, the carrier filter was removed from the fluorescent dye solution after a contact reaction time deemed appropriate for detection.
また，一方では．一旦発光したものが時間と共に薄くな
るという，いわゆる褪色現象が生し，検出感度が低下し
てしまう。Also, on the other hand. A so-called fading phenomenon occurs in which the emitted light becomes fainter over time, resulting in a decrease in detection sensitivity.
また，従来は前記のごとく２ハイブリノド形成時に前記
反応試薬液をパッケージに入れて反応させるために１高
価な反応試薬液を多量必要としていた．
本発明はかかる従来の問題点に鑑み，ＤＮＡ断片等から
発せられる光信号等を順次リアルタイムで検出すること
ができ．かつ反応試薬液量も少なくすることができる，
検出感度に優れた，ＤＮＡ断片等の検出システムを提供
しようとするものである．
〔課題の解決手段〕
本発明において，ＤＮＡ断片等の被検出物が固定された
担体フィルターを取り付けるローターと，反応試薬液を
入れて該反応試薬液中に上記ローターを浸漬させるため
の反応槽と，上記ローターを反応槽内において回転させ
るための駆動源と．上記ＤＮＡ断片等の被検出物から発
せられる光１放射線等の信号を検出する検出器と，該検
出器からの信号を画像としてディスプレイ上に表示する
ための画像表示器とよりなることを特徴とするＤＮＡ断
片等の検出システムにある．
しかして，上記被検出物としては，ＤＮＡ，ＲＮＡ，タ
ンパク等を制限酵素で切断したＤＮＡ断片，ＲＮＡ断片
．タンパクなどがある．本発明において最も注目すべき
ことは，前記担体フィルターをローターの外周に貼着し
，該担体フィルターを反応槽中の反応試薬液．更には洗
浄液に浸漬すると共にローターを回転させ，担体フィル
ター上の被検出物から発せられる光信号等を順次検出器
により検出するようにしたことにある．上記ローターは
円筒状の外周面を有し．電気モ−ター等の駆動源によっ
て回転させられる。反応槽は，反応試薬液．洗浄液を入
れると共に，上記ローター上の担体フィルターを浸漬す
るためのものである．上記洗浄液は．通常３〜５種類を
用いる．そのため，反応槽は，上記反応試薬液，洗浄液
を供給又は排出するための液供給口を有することが望ま
しい（実施例参照）。Furthermore, as mentioned above, in the past, a large amount of an expensive reaction reagent solution was required in order to put the reaction reagent solution into a package and allow the reaction to occur when forming two hybrid nodes. In view of these conventional problems, the present invention is capable of sequentially detecting optical signals etc. emitted from DNA fragments etc. in real time. In addition, the amount of reaction reagent liquid can be reduced.
The aim is to provide a detection system for DNA fragments, etc. with excellent detection sensitivity. [Means for Solving the Problems] The present invention includes a rotor to which a carrier filter on which an analyte such as a DNA fragment is immobilized is attached, and a reaction tank in which a reaction reagent solution is placed and the rotor is immersed in the reaction reagent solution. , a drive source for rotating the rotor in the reaction tank; It is characterized by comprising a detector that detects a signal such as light 1 radiation emitted from the object to be detected such as the DNA fragment, and an image display device that displays the signal from the detector as an image on a display. This is a system for detecting DNA fragments, etc. The above-mentioned substances to be detected include DNA fragments and RNA fragments obtained by cutting DNA, RNA, proteins, etc. with restriction enzymes. There are proteins, etc. The most noteworthy feature of the present invention is that the carrier filter is attached to the outer periphery of the rotor, and the carrier filter is attached to the reaction reagent solution in the reaction tank. Furthermore, the rotor is rotated while being immersed in a cleaning solution, and the optical signals etc. emitted from the detection object on the carrier filter are sequentially detected by a detector. The above rotor has a cylindrical outer peripheral surface. It is rotated by a drive source such as an electric motor. The reaction tank contains the reaction reagent solution. This is to contain the cleaning solution and also to immerse the carrier filter on the rotor. The above cleaning solution is. Usually 3 to 5 types are used. Therefore, it is desirable that the reaction tank has a liquid supply port for supplying or discharging the reaction reagent liquid and cleaning liquid (see Examples).
また，検出器は，ＤＮＡ断片等から発せられる光，放射
線等の信号を検出するセンサーである。Further, the detector is a sensor that detects signals such as light and radiation emitted from DNA fragments and the like.
該検出器は，反応槽中の担体フィルターに向けて反応槽
の背壁等に設ける。The detector is installed on the back wall of the reaction tank, facing the carrier filter in the reaction tank.
また，画像表示器は上記検出信号に基づいて担体フィル
ター上のＤＮＡ断片の位置をディスプレイ上に表示する
ものである。また，該画像表示器には，必要に応して，
記録装置を設ける。更に上記検出器と画像表示器との間
には，検出器が検出した信号をデータとして取り扱うた
めに数値に変換する画像取込部と，そのデータを処理す
るデータ処理部を設ける。Further, the image display device displays the position of the DNA fragment on the carrier filter on a display based on the detection signal. In addition, the image display may include, if necessary,
A recording device will be provided. Further, between the detector and the image display, there is provided an image capture section that converts the signal detected by the detector into a numerical value in order to handle it as data, and a data processing section that processes the data.
また．上記検出器の近傍には，担体フィルター上のＤＮ
Ａ断片等を発光させるための光源と．該光を前記担体フ
ィルター上に向けて照射するための送光器を設けること
が好ましい．即ち．標識が生物発光，Ｒｆの場合には上
記送光器は必要としないが，蛍光色素や酵素の場合には
送光器を必要とする．
〔作 用〕
本発明の検出システムにおいては，まずＤＮＡ断片等の
被検出物が固定された担体フィルターをローターの外周
に取り付ける．このとき２担体フィルターは，ＤＮＡ断
片の分両方向がローターを周回するように配置する．次
いで，該ローターを．反応試薬液が入っている反応槽内
に装着すると共に回転させる．
この回転により，担体フィルターは反応試薬液に浸漬さ
れ．担体フィルター上のＤＮＡ断片が反応試薬液と反応
してハイブリダイゼーシゴンを起こす．ハイブリッド形
威後，反応試薬液を排出し，洗浄液を入れ．担体フィル
ター上の相補結合していないＤＮＡプロープを洗い落と
す．このようにして，３〜４種類の洗浄液で洗浄操作を
行なう．全洗浄を終了した後．検出用の酵素．蛍光色素
が入った溶液を入れ，ＤＮＡ断片を発光させる．そして
，検出器により上記発光等の信号を検出し画像表示部に
表示させる。なお，上記操作の間，ローターは全て回転
している．
それ故，担体フィルター上のＤＮＡ断片より発せられる
呈色光等の信号は常時連続して検出される．そのため，
蛍光色素を加えた後の発光状態が時間と共に強くなって
いく過程の状態も全て検出でき，いわゆるリアルタイム
処理の検出が行われる．それ故，最適発光状態をキャッ
チすることができ，正確なＤＮＡ断片検出を行なうこと
ができる．
また，担体フィルター上のＤＮＡ断片から発せられる個
々の信号は，ローターの回転が１周するごとに検出器で
その都度検出されるため，全てのＤＮＡ断片からの信号
が多数回検出される。それ故，これら信号を積算すれば
信号とノイズの部分の差が広がり（Ｓ／Ｎ比の向上），
検出感度が向上ずる．
また．反応槽中の反応試薬液は少量で済むのでコスト低
減となる。Also. Near the above detector, there is a DN on a carrier filter.
A light source for emitting light from fragment A, etc. It is preferable to provide a light transmitter for irradiating the light onto the carrier filter. That is. If the label is bioluminescence or Rf, the light transmitter described above is not required, but if the label is a fluorescent dye or enzyme, a light transmitter is required. [Function] In the detection system of the present invention, first, a carrier filter on which a detection object such as a DNA fragment is immobilized is attached to the outer periphery of the rotor. At this time, the two-carrier filter is arranged so that both directions of the DNA fragments revolve around the rotor. Next, the rotor. Place it in the reaction tank containing the reaction reagent solution and rotate it. Due to this rotation, the carrier filter is immersed in the reaction reagent solution. The DNA fragments on the carrier filter react with the reaction reagent solution to cause hybridization. After hybridization, drain the reaction reagent solution and add washing solution. Wash off non-complementarily bound DNA probes on the carrier filter. In this way, cleaning operations are performed using three to four types of cleaning solutions. After completing all cleaning. Enzyme for detection. Add a solution containing a fluorescent dye to make the DNA fragments emit light. Then, a detector detects the light emission signal and displays it on the image display section. Note that all the rotors are rotating during the above operation. Therefore, signals such as colored light emitted from the DNA fragments on the carrier filter are continuously detected at all times. Therefore,
It is also possible to detect all the states in which the luminescence becomes stronger over time after adding the fluorescent dye, resulting in so-called real-time detection. Therefore, it is possible to detect the optimal luminescent state and perform accurate DNA fragment detection. Furthermore, each signal emitted from the DNA fragments on the carrier filter is detected by the detector each time the rotor rotates once, so the signals from all the DNA fragments are detected many times. Therefore, by integrating these signals, the difference between the signal and noise increases (improving the S/N ratio),
Detection sensitivity improves. Also. Since only a small amount of reaction reagent solution is required in the reaction tank, costs are reduced.
したがって，本発明によれば，ＤＮＡ断片等から発せら
れる光信号等を反応及び洗浄中にリアルタイムで検出す
ることができ，また反応試薬液やＤＮＡブローブ等が少
なくなるうえに，ロータ回転により反応試薬液の撹拌と
共に検体間に高精度で高感度な検出システムを提供する
ことができる．〔実施例〕
本発明の実施例にかかる，ＤＮＡ断片の検出システムに
つき，第１図ないし第５図を用いて説明する．
本例の検出システムは．第１図にその全体を示すごとく
．担体フィルター２を取り付けるローター１と，反応試
薬液３０，或いは洗浄液を入れる反応槽３と，検出器４
５と．画像表示器としての画像表示部４８と，光源４１
とよりなる．上記ローター■は．第２図に示すごとく，
円筒状の本体１０と，その両端に設けたシャフト１１と
よりなる。該ローター１の外周には，その円周に沿って
担体フィルター２を，ＤＮＡ断片２１が分両方向となる
ように貼着する。Therefore, according to the present invention, optical signals etc. emitted from DNA fragments, etc. can be detected in real time during reaction and washing, and the amount of reaction reagent solution, DNA probe, etc. can be reduced, and the reaction reagent can be removed by rotating the rotor. It is possible to provide a highly accurate and sensitive detection system between samples while stirring the liquid. [Example] A DNA fragment detection system according to an example of the present invention will be explained using FIGS. 1 to 5. The detection system in this example is. The entire structure is shown in Figure 1. A rotor 1 to which a carrier filter 2 is attached, a reaction tank 3 containing a reaction reagent solution 30 or a cleaning solution, and a detector 4
5 and. An image display unit 48 as an image display and a light source 41
It depends. The rotor ■ above is. As shown in Figure 2,
It consists of a cylindrical main body 10 and shafts 11 provided at both ends thereof. A carrier filter 2 is attached to the outer periphery of the rotor 1 along its circumference so that the DNA fragments 21 are oriented in both directions.
上記反応槽３は，第３図〜第５図に示すごとく反応試薬
液３０を入れる底皿部３１と．蓋部３２とよりなり，両
者はヒンジ３２６で結合されている．また．蓋部３２は
，取手３２５を有する．更に，上記底皿部３１の下方に
は，反応試薬液３０等を供給，排出するための液供給口
３６を有する．また，底皿部３１，蓋部３２は半円状の
空洞３１１，３２１を有する（第５図）。また，底皿部
３１の一端には．ローター１の回転用のモータ１２を．
他端にはローター１０の軸受３７を設ける．また，第１
図に示すごとく，底皿部３１内には，温度コントローラ
３５１に接続した温度制御用モジュール３５を設ける．
次に，第４図，第５図に示すごとく，上記反応槽３の背
面部には．蓋３２と底皿部３１との間に抹出器４５を設
ける．該検出器４５は，第２図に示すごとく．ローター
ｌＯ上の担体フィルター２に向けて配設されている．該
検出器４５は，担体フィルター２の全幅に及ぶ長さを有
し，その先端部に送光器４１１と画像センサ４５１とを
有している．
しかして，第１図に示すごとく，上記送光器４１ｌは光
′ａ４１に光ファイバーにより接続し，一方画像センサ
４５１は，画像取込部４６，データ処理部４７．画像表
示部４８に電気的に接続する．次に，上記装置を用いて
検出する方法につき説明する．
まず，第１図に示すごとく，反応槽３の底皿部３２内に
液供給口３６より，反応試薬液溶液３０を入れる．該反
応試薬液３０は，前記ハイブリダイゼーシゴン反応を行
なう反応試薬液である．この反応試薬液中には，目的と
する遺伝子に対応するＤＮＡと．後述する蛍光色素と反
応する中間物質が含まれている．次に．温度制橢モジュ
ール３５により，反応試薬液３０を一定温度（例えば．
６５゜Ｃ，又は４２゜Ｃなど）に加熱保持する．その後
，Ｍ部３２を開げ３第ｌ図〜第３図に示すごとく担体フ
ィルター２を貼着したローター１を．その両端のシャフ
ト１１．１１がモータ１２．軸受３７と連結するよう底
皿部３２上に置く．これにより．ローター１と担体フィ
ルター２の下方が反応試薬液３０に浸漬される．
次いで，モータ１２によりローターｌを回転させる．こ
れにより，担体フィルター２上のＤＮＡ断片２１が反応
試薬液３０と接触し，反応試薬液中の前記ＤＮＡ及び中
間物質とＤＮＡ断片２１との間でハイブリダイゼーショ
ン反応が起きる．そして．この反応が終わると洗浄工程
を行なう。この洗浄工程では．まず液供給口３６より，
上記のハイプリダイゼーション用の反応試薬液を排出し
，新たに洗浄液を入れる．この洗浄は，塩濃度を徐々に
薄くシたものを用いて．４回行なう．上記各洗浄は，約
２時間当て行なう。The reaction tank 3 has a bottom plate part 31 into which a reaction reagent solution 30 is placed, as shown in FIGS. 3 to 5. The lid part 32 is connected to the lid part 32 by a hinge 326. Also. The lid portion 32 has a handle 325. Furthermore, a liquid supply port 36 is provided below the bottom plate 31 for supplying and discharging the reaction reagent liquid 30 and the like. Moreover, the bottom plate part 31 and the lid part 32 have semicircular cavities 311 and 321 (FIG. 5). Moreover, at one end of the bottom plate part 31. A motor 12 for rotating the rotor 1.
A bearing 37 for the rotor 10 is provided at the other end. Also, the first
As shown in the figure, a temperature control module 35 connected to a temperature controller 351 is provided inside the bottom plate 31. Next, as shown in FIGS. 4 and 5, on the back side of the reaction tank 3. An obliterator 45 is provided between the lid 32 and the bottom plate part 31. The detector 45 is as shown in FIG. It is arranged toward the carrier filter 2 on the rotor IO. The detector 45 has a length that spans the entire width of the carrier filter 2, and has a light transmitter 411 and an image sensor 451 at its tip. As shown in FIG. 1, the light transmitter 41l is connected to the light 'a41 by an optical fiber, while the image sensor 451 is connected to the image capture section 46, the data processing section 47. It is electrically connected to the image display section 48. Next, we will explain the detection method using the above device. First, as shown in FIG. 1, a reaction reagent solution 30 is poured into the bottom pan 32 of the reaction tank 3 through the liquid supply port 36. The reaction reagent solution 30 is a reaction reagent solution for carrying out the hybridization reaction. This reaction reagent solution contains DNA corresponding to the gene of interest. It contains an intermediate substance that reacts with the fluorescent dye described below. next. The temperature control module 35 keeps the reaction reagent solution 30 at a constant temperature (for example.
Heat and hold at 65°C, 42°C, etc.). Thereafter, the M section 32 is opened and the rotor 1 with the carrier filter 2 attached thereto is inserted as shown in FIGS. The shafts 11.11 at both ends are the motors 12. Place it on the bottom plate 32 so as to connect it with the bearing 37. Due to this. The lower part of the rotor 1 and carrier filter 2 is immersed in the reaction reagent solution 30. Next, the rotor l is rotated by the motor 12. As a result, the DNA fragments 21 on the carrier filter 2 come into contact with the reaction reagent solution 30, and a hybridization reaction occurs between the DNA fragments 21 and the DNA and intermediate substances in the reaction reagent solution. and. After this reaction is completed, a washing step is performed. In this cleaning process. First, from the liquid supply port 36,
Drain the above reaction reagent solution for hybridization and add new washing solution. For this cleaning, use a solution that gradually reduces the salt concentration. Do this 4 times. Each of the above washings is carried out for about 2 hours.
そして，上記，洗浄を全て終わってから，検出用の蛍光
色素の入った溶液を入れる．これにより上記蛍光色素と
前記ハイブリダイゼーシゴン時の中間物質とが反応し，
発光可能となる，この間，ローター１はモータ１２によ
って常時回転している。After completing all of the washing steps mentioned above, add a solution containing a fluorescent dye for detection. As a result, the fluorescent dye and the intermediate substance during hybridization react,
During this period, the rotor 1 is constantly rotated by the motor 12 during which light can be emitted.
次いで．光源４１より呈色発光し易い波長の光を送光器
４１１より，阻体フィルター２に向けて当てる。これに
より．担体フィルター２上のＤＮＡ断片２１と結合した
前記蛍光色素がこの光によって励起され．発光色素が光
を出す．
そこで，この光を，検出器４５の画像センサー４５１に
より検出し，その情報を画像取込部４６に送る．画像取
込部４６では上記光信号の情報をデータに変換し，更に
データ処理部４７で画像として処理し，ローター１の回
転によって担体フィルター２から順次繰り返し送られて
くるデータを積算する。そして，画像表示部４８のディ
スプレイ上において，ＤＮＡ断片２１の状態が表示され
る．
しかして，上記のローター１の回転，検出ないし表示操
作は，前記の反応開始時から．検出終了まで常時行われ
ている．そのため，標識としてＲ【を用いた場合には画
像表示部には，この間の全ての情報，即ち反応試薬液と
の反応過程におけるＤＮＡ断片２ｌの状態が連続して表
示され，更には記録される．一方．上記のごとく洗浄後
に蛍光物質を加えて発光させる場合は，この後に連続し
て上記検出がなされる．また．ハイブリダイゼーション
当初より蛍光を出す物質を結合させておいた場合には，
前記ハイプリダイゼーション中及びその洗浄後全てのと
きに上記検出ができる。Next. A light source 41 directs light having a wavelength that is likely to emit colored light from a light transmitter 411 toward the blocking filter 2 . Due to this. The fluorescent dye bound to the DNA fragment 21 on the carrier filter 2 is excited by this light. Luminescent pigments emit light. Therefore, this light is detected by the image sensor 451 of the detector 45 and the information is sent to the image capturing section 46. The image capturing section 46 converts the information of the optical signal into data, which is further processed as an image by the data processing section 47, and the data sequentially and repeatedly sent from the carrier filter 2 as the rotor 1 rotates is integrated. Then, the state of the DNA fragment 21 is displayed on the display of the image display section 48. Therefore, the rotation of the rotor 1 and the detection or display operations described above start from the start of the reaction. This process continues until the detection is complete. Therefore, when R is used as a label, all the information during this time, that is, the state of the DNA fragment 2l during the reaction process with the reaction reagent solution, is continuously displayed and further recorded. ．． on the other hand. If a fluorescent substance is added after washing to emit light as described above, then the above detection is performed continuously. Also. If a fluorescent substance is bound from the beginning of hybridization,
The above detection can be performed during the hybridization and after the washing.
それ故．担体フィルター２上の状態をリアルタイムで処
理することができる．即ち．上記発光は，前記のごとく
，蛍光色素を入れた後には徐々に発光力を強めていくが
．本システムはこの変化過程を逐次検出できるためリア
ルタイムで検出できる．なお，従来は，前記のごとく，
蛍光色素溶液に担体フィルターを入れた後，適当な時間
後に蛍光色素溶液から担体フィルターを出して検出して
いたため，発光が不充分又は多すぎてノイズを拾ってし
まい検出精度が悪かった．
また，本例においては．ローターが■回転する毎に，担
体フィルター上の情報が再度得られるので，何度も同じ
情報を繰り返し入手することができ，検出感度を上げる
ことができる．
また，反応試薬液３０は，反応槽３の底部に少量入れ，
これに回転する担体フィルター２を何度も浸漬して使用
することができるので，その量を少量とすることができ
る．Therefore. The status on the carrier filter 2 can be processed in real time. That is. As mentioned above, the luminescence power gradually increases after adding the fluorescent dye. This system can detect this change process sequentially, so it can be detected in real time. Note that conventionally, as mentioned above,
Since the carrier filter was placed in the fluorescent dye solution and then removed from the fluorescent dye solution after a certain period of time for detection, the luminescence was insufficient or too much and noise was picked up, resulting in poor detection accuracy. Also, in this example. Every time the rotor rotates, the information on the carrier filter is obtained again, so the same information can be obtained over and over again, increasing detection sensitivity. In addition, a small amount of the reaction reagent solution 30 is added to the bottom of the reaction tank 3.
Since the rotating carrier filter 2 can be immersed in this water many times, the amount of water used can be reduced.
第１図〜第５図は実施例にかかる検出システムを示し第
１図はその全体図，第２図はローター及び検出器の斜視
図，第３図は反応槽の一部断面正面図，第４図は反応槽
の背面図，第５図は蓋を開けた状態の反応槽の平面図で
ある．
ｌ
２．
２ １．
３．
３ １．
４ ５、
ローター
担体フィルター
ＤＮＡ断片，
反応槽，
底皿部，
検出器，
第１図
第２図
第４
図
第５帯Figures 1 to 5 show the detection system according to the embodiment. Figure 1 is an overall view of the system, Figure 2 is a perspective view of the rotor and detector, Figure 3 is a partially sectional front view of the reaction tank, and Figure 3 is a partial cross-sectional front view of the reaction tank. Figure 4 is a rear view of the reaction tank, and Figure 5 is a plan view of the reaction tank with the lid open. l 2. 2 1. 3. 3 1. 4 5. Rotor carrier filter DNA fragment, reaction tank, bottom plate, detector, Fig. 1 Fig. 2 Fig. 4 Fig. 5 band
Claims (1)
取り付けるローターと、反応試薬液を入れて該反応試薬
液中に上記ローターを浸漬させるための反応槽と、上記
ローターを反応槽内において回転させるための駆動源と
、上記ＤＮＡ断片等の被検出物から発せられる光、放射
線等の信号を検出する検出器と、該検出器からの信号を
画像としてディスプレイ上に表示するための画像表示器
とよりなることを特徴とするＤＮＡ断片等の検出システ
ム。A rotor to which a carrier filter on which an object to be detected such as a DNA fragment is immobilized is attached, a reaction tank in which a reaction reagent solution is placed and the rotor is immersed in the reaction reagent solution, and the rotor is rotated within the reaction tank. a detector for detecting signals such as light and radiation emitted from the detected object such as the DNA fragment; and an image display for displaying the signal from the detector as an image on a display. A detection system for DNA fragments, etc., characterized by the following.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7733489A JPH0354463A (en) | 1989-03-28 | 1989-03-28 | Apparatus for detecting dna segment or the like |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7733489A JPH0354463A (en) | 1989-03-28 | 1989-03-28 | Apparatus for detecting dna segment or the like |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0354463A true JPH0354463A (en) | 1991-03-08 |
Family
ID=13631027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7733489A Pending JPH0354463A (en) | 1989-03-28 | 1989-03-28 | Apparatus for detecting dna segment or the like |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0354463A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009156285A (en) * | 2007-12-25 | 2009-07-16 | Daifuku Co Ltd | Connection structure for bar-shaped member and attached plate part |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5332093A (en) * | 1976-09-06 | 1978-03-25 | Olympus Optical Co Ltd | Automatic electrophoresis apparatus |
| JPS59126253A (en) * | 1983-01-08 | 1984-07-20 | Fuji Photo Film Co Ltd | Signal processing in autoradiography |
| JPS6240556B2 (en) * | 1982-10-29 | 1987-08-28 | Carrier Corp |
-
1989
- 1989-03-28 JP JP7733489A patent/JPH0354463A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5332093A (en) * | 1976-09-06 | 1978-03-25 | Olympus Optical Co Ltd | Automatic electrophoresis apparatus |
| JPS6240556B2 (en) * | 1982-10-29 | 1987-08-28 | Carrier Corp | |
| JPS59126253A (en) * | 1983-01-08 | 1984-07-20 | Fuji Photo Film Co Ltd | Signal processing in autoradiography |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009156285A (en) * | 2007-12-25 | 2009-07-16 | Daifuku Co Ltd | Connection structure for bar-shaped member and attached plate part |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0891546B1 (en) | Automatic diagnostic apparatus | |
| D'Orazio | Biosensors in clinical chemistry | |
| CN1764834A (en) | Biosensor and biosensor measuring device and method | |
| CN101213447B (en) | Blo drive apparatus, and assay method using the same | |
| US4997627A (en) | Sample analysis | |
| US9434987B2 (en) | Lab on chip cartridge | |
| US6605471B1 (en) | Method and system for determining at least one parameter of at least one sample of a physiological liquid, a holder and a test device | |
| US4652137A (en) | Centrifugal analyzer | |
| US20150010993A1 (en) | System for conducting the identification of bacteria in urine | |
| US20120277553A1 (en) | Spectrophotometric measurement in color-based biochemical and immunological assays | |
| JP4061816B2 (en) | Biosensor | |
| US20100227384A1 (en) | Portable Genomic Analyzer | |
| CN110241008A (en) | A kind of device for extracting and detecting for large biological molecule | |
| GB1601222A (en) | Method and apparatus for measurement of the aggregation rate of red blood corpuscles suspended in blood | |
| JP3090692B2 (en) | Enzyme reaction measuring method and its measuring device | |
| JPS62185148A (en) | Method for optically determining enzyme activity by catalytic reaction of sample and system for executing said method | |
| JPH0354463A (en) | Apparatus for detecting dna segment or the like | |
| JP3055342B2 (en) | DNA probe automatic measuring device and method for measuring nucleic acid in sample | |
| JPH05172828A (en) | Automatic analyser | |
| CN108467726A (en) | It is a kind of quantitatively to detect near infrared fluorescent probe of endogenous hydrogen peroxide and preparation method thereof, application for ratio | |
| CA3062304A1 (en) | Biosensors produced from enzymes with reduced solubility and methods of production and use thereof | |
| JP3531196B2 (en) | Method and apparatus for detecting a substance by a luminescence reaction on a porous membrane | |
| JP3036791B2 (en) | Nucleic acid detection method and nucleic acid detection container used therefor | |
| JP2000083647A (en) | Gene detector and method therefor | |
| JPH1194743A (en) | Reader for fluorescent pattern |