JPH0568596A - Detection of dna multiplied by polymerase chain reaction - Google Patents
Detection of dna multiplied by polymerase chain reactionInfo
- Publication number
- JPH0568596A JPH0568596A JP19987991A JP19987991A JPH0568596A JP H0568596 A JPH0568596 A JP H0568596A JP 19987991 A JP19987991 A JP 19987991A JP 19987991 A JP19987991 A JP 19987991A JP H0568596 A JPH0568596 A JP H0568596A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- pcr
- polymerase chain
- chain reaction
- reaction
- 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
- 238000003752 polymerase chain reaction Methods 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title claims description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000000126 substance Substances 0.000 claims abstract description 21
- 241000700605 Viruses Species 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 2
- 239000002246 antineoplastic agent Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims description 2
- 238000012758 nuclear staining Methods 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 claims 1
- 230000000007 visual effect Effects 0.000 abstract description 14
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 abstract description 10
- 229960005542 ethidium bromide Drugs 0.000 abstract description 10
- 239000000243 solution Substances 0.000 description 21
- 239000000047 product Substances 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 10
- 208000005176 Hepatitis C Diseases 0.000 description 8
- 238000012360 testing method Methods 0.000 description 6
- 238000010979 pH adjustment Methods 0.000 description 5
- 241000711549 Hepacivirus C Species 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000005284 excitation Effects 0.000 description 3
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- VIBDVOOELVZGDU-UHFFFAOYSA-N 4-(1h-indol-2-yl)benzene-1,3-dicarboximidamide Chemical compound NC(=N)C1=CC(C(=N)N)=CC=C1C1=CC2=CC=CC=C2N1 VIBDVOOELVZGDU-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- CFCUWKMKBJTWLW-UHFFFAOYSA-N deoliosyl-3C-alpha-L-digitoxosyl-MTM Natural products CC=1C(O)=C2C(O)=C3C(=O)C(OC4OC(C)C(O)C(OC5OC(C)C(O)C(OC6OC(C)C(O)C(C)(O)C6)C5)C4)C(C(OC)C(=O)C(O)C(C)O)CC3=CC2=CC=1OC(OC(C)C1O)CC1OC1CC(O)C(O)C(C)O1 CFCUWKMKBJTWLW-UHFFFAOYSA-N 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- CFCUWKMKBJTWLW-BKHRDMLASA-N mithramycin Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@H](O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@@H](O)[C@H](O)[C@@H](C)O1 CFCUWKMKBJTWLW-BKHRDMLASA-N 0.000 description 1
- 229960003171 plicamycin Drugs 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- UKOBAUFLOGFCMV-UHFFFAOYSA-N quinacrine mustard Chemical compound C1=C(Cl)C=CC2=C(NC(C)CCCN(CCCl)CCCl)C3=CC(OC)=CC=C3N=C21 UKOBAUFLOGFCMV-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は微量の DNA 又は RNA を
鋳型としたものを用い、ポリメラーゼ連鎖反応 [Polyme
rase Chain Reaction (以下、「PCR」と略記する] によ
り、その産物である DNA を増幅させ、この増幅した DN
A を蛍光物質と結合させて蛍光の有無から判定する DNA
の検出法に係る。[Field of Industrial Application] The present invention uses polymerase chain reaction [Polyme
The product DNA is amplified by rase Chain Reaction (hereinafter abbreviated as “PCR”), and the amplified DN is amplified.
DNA that binds A with a fluorescent substance and determines from the presence or absence of fluorescence
Related to the detection method of.
【０００２】[0002]
【従来の技術】PCR 法は、増幅させたい DNA 領域を挟
む 2 種類のプライマーを用いて DNAポリメラーゼによ
る DNA 合成反応を繰り返すことによって、目的とする
DNA 断片を比較的短時間で数 10 万倍乃至 100 万倍以
上に増幅させる方法である。従ってPCR 法は、原理的に
は、DNA が 1 コピーあれば、これを増幅させて極めて
多数個になし得る方法であるためにウイルスの検出、癌
の診断、遺伝病の診断等の臨床面での応用が期待されて
いる。2. Description of the Related Art The PCR method is aimed at by repeating a DNA synthesis reaction by a DNA polymerase using two kinds of primers flanking a DNA region to be amplified.
This is a method for amplifying a DNA fragment several hundred thousand times to one million times or more in a relatively short time. Therefore, the PCR method is, in principle, a method in which if one copy of DNA is amplified, it can be amplified to an extremely large number, so that it is clinically applicable to virus detection, cancer diagnosis, genetic disease diagnosis, etc. Is expected to be applied.
【０００３】しかしながら、PCR 産物である DNA の検
出においては、一般にアガロース又はポリアクリルアミ
ドゲル電気泳動と、それに続く臭化エチジウム染色、更
に UV 照射による反応産物の観察と写真撮影を行う必要
がある。従って、PCR 法を利用する従来の DNA 検出法
は、操作が煩雑で所要時間も長いので処理検体の数が限
られてしまい、その結果、多数の検体の処理が要求され
る一般の病院や臨床検査室では実施が困難な状況にあ
る。However, in the detection of DNA which is a PCR product, it is generally necessary to perform agarose or polyacrylamide gel electrophoresis, followed by ethidium bromide staining, and observation and photography of the reaction product by UV irradiation. Therefore, the conventional DNA detection method using the PCR method is complicated in operation and takes a long time, so that the number of samples to be processed is limited, and as a result, general hospitals and clinical clinics requiring processing of many samples are required. It is difficult to carry out in the laboratory.
【０００４】このような状況に鑑み、本発明者等は、こ
れまでに PCR で増幅した DNA の簡単な検出方法とし
て、DNA に親和性を有する蛍光物質を用いることにより
簡便であり多検体を短時間で処理できる方法を開発して
特許出願した (特願平 3 - 26477)。この方法を用いれ
ば、例えば C 型肝炎ウイルスを検出する場合に PCR 後
の DNA の検出において蛍光強度を測定するだけなの
で、100 人の患者血清を検体とする検査が僅か 1 時間
で完了する。このことは、従来の電気泳動による検出法
と比較する場合に、所要時間が約 1/6 に短縮すること
を意味している。In view of such a situation, the present inventors have used a fluorescent substance having an affinity for DNA as a simple method for detecting DNA amplified by PCR so far, which is simple and short for many samples. We developed a method that can be processed in time and applied for a patent (Japanese Patent Application No. 3-26477). Using this method, for example, when detecting hepatitis C virus, only the fluorescence intensity is measured in the detection of DNA after PCR, so that the test using 100 patient sera can be completed in only 1 hour. This means that the time required is reduced to about 1/6 when compared with the conventional electrophoresis detection method.
【０００５】尚、PCR を利用する DNA の検出法は、PCR
が極めて高感度な反応であるために、DNA に僅かなコン
タミネーションがあれば、本来「陰性」の検体が見掛け
上「陽性」と誤判定されることが大きな問題となってい
る。これに関連して言及するに、従来の方法では、PCR
産物の検出の際には必ず反応チューブより反応液を取り
出す必要がある。この際に、PCR 産物である増幅された
DNA が極く僅かでも飛散したり、実験器具や測定者の
手・衣服に付着してキャリーオーバーされることが上記
のコンタミネーションを引き起こす原因となる。従っ
て、PCR を利用する診断法は、有用であることが明らか
であるにも拘らず、PCR 産物のコンタミネーションが障
害となって取扱いが難しいために、一般に普及していな
いのが実状である。A method of detecting DNA using PCR is PCR
Since it is an extremely sensitive reaction, if there is a slight amount of contamination in the DNA, it is a big problem that an originally “negative” sample is erroneously judged to be “positive”. In this regard, the conventional method is PCR
When detecting products, it is necessary to take out the reaction solution from the reaction tube. At this time, the PCR product amplified
Even the slightest amount of DNA scatters, or adheres to laboratory equipment or the hands / clothes of the measurer and is carried over, which causes the above contamination. Therefore, although it is clear that the diagnostic method using PCR is useful, it is difficult to handle because the contamination of the PCR product is an obstacle and it is difficult to handle it.
【０００６】[0006]
【発明が解決しようとする課題】現在迄の処、PCR 技術
を利用する DNA の検出法であって、PCR 後に反応チュ
ーブから反応液を取り出さずに、PCR 産物である DNA
の存否を判定、殊に目視で判定できる DNA の検出法は
確立されるに至っていない。[Problems to be Solved by the Invention] So far, there has been a method for detecting DNA using PCR technology, which is a PCR product without removing the reaction solution from the reaction tube after PCR.
A method for detecting DNA, which can determine the presence or absence of DNA, particularly by visual inspection, has not been established.
【０００７】本発明は従来技術における既述の問題点乃
至困難を踏まえてなされたものであり、本発明の課題
は、PCR で増幅した DNA の検出において、PCR 後の反
応液を反応チューブの外に取り出さずに且つ目視観察に
よっても DNA の存否を判定できる、簡便にして正確な
DNA の検出法を提供することにある。The present invention has been made in view of the above-mentioned problems and difficulties in the prior art, and an object of the present invention is to detect a DNA amplified by PCR from the reaction solution outside the reaction tube. The presence and absence of DNA can be determined by visual observation without removing
It is to provide a method for detecting DNA.
【０００８】更に、この目視観察による判定法は、DNA
と蛍光物質との結合によって生じる蛍光を利用する方法
であるので、この際に実験者の主観に左右されないよう
に「陽性」検体と「陰性」検体との蛍光強度の差を 5
倍以上に設定する必要性がある。そこで本発明の次の課
題は、殊に「陰性」検体に関する蛍光強度をできるだけ
低く抑える技術を提供することにある。[0008] Furthermore, the determination method by this visual observation is DNA
Since this method utilizes the fluorescence generated by the binding of the fluorescent substance with the fluorescent substance, the difference in the fluorescence intensity between the “positive” sample and the “negative” sample should not be influenced by the subjectivity of the experimenter.
It is necessary to set it more than twice. Therefore, the next object of the present invention is to provide a technique for suppressing the fluorescence intensity of “negative” specimens as low as possible.
【０００９】[0009]
【課題を解決するための手段及び作用】本発明者等は、
PCR を利用する DNA の検出法について鋭意検討を行な
った結果、DNA に親和性を有する蛍光物質を PCR 後の
反応チューブに加えた後、反応チューブより反応液を取
り出すことなく、上記の蛍光物質に特異的な波長の励起
光を反応チューブに直接照射して、反応液の蛍光強度を
目視観察することにより簡単に DNA の存否を判定し、
延いては「陽性」検体と「陰性」検体とを区別し得るこ
とを見い出した。本発明による DNA の検出法におい
て、当該 DNA に親和性を有する蛍光物質としては、抗
生物質、抗癌剤、DNA 及び RNA の標識用蛍光試薬並び
に核染色用の蛍光色素等を用いることができる。これら
の蛍光物質としては、例えばミトラマイシン、アクリジ
ンオレンジ、臭化エチジウム、4',6'-ジアミジノ-2-フ
ェニルインドール、キナクリンマスタード等を例示する
ことができ、これらの内でも殊に臭化エチジウムを用い
るのが好ましい。Means and Actions for Solving the Problems The present inventors have
As a result of diligent studies on the method of detecting DNA using PCR, after adding a fluorescent substance having an affinity for DNA to the reaction tube after PCR, the above fluorescent substance was added without removing the reaction solution from the reaction tube. The presence or absence of DNA can be easily determined by directly irradiating the reaction tube with excitation light of a specific wavelength and visually observing the fluorescence intensity of the reaction solution.
Furthermore, it has been found that it is possible to distinguish between "positive" and "negative" specimens. In the method for detecting DNA according to the present invention, as the fluorescent substance having an affinity for the DNA, antibiotics, anticancer agents, fluorescent reagents for labeling DNA and RNA, fluorescent dyes for nuclear staining and the like can be used. Examples of these fluorescent substances include mithramycin, acridine orange, ethidium bromide, 4 ′, 6′-diamidino-2-phenylindole, quinacrine mustard, and the like. Among these, ethidium bromide is particularly preferable. Is preferably used.
【００１０】本発明者等は、「陰性」検体の蛍光強度を
できるだけ低く抑える技術について更に検討を行った結
果、先ず「陰性」検体の蛍光は PCR に用いるプライマ
ーによるものであることを見い出した。次に、このプラ
イマーによる非特異的な蛍光の発生は PCR 後の反応溶
液の pH を調整することにより抑制し得ることを見い出
した。即ち、反応溶液の pH を 10 - 12 に保てば、
「陰性」検体中のプライマーによる蛍光は目視観察では
全く感知できなくのである。一方、この pH 条件下にお
いて「陽性」検体中に存在し且つ PCR で増幅した DNA
は二本鎖を形成することができ、従って蛍光物質と特異
的に反応して蛍光を発するので、「陰性」検体と「陽
性」検体とは目視観察によっても確実に区別することが
可能になった。As a result of further studies on the technique for suppressing the fluorescence intensity of the “negative” sample as low as possible, the present inventors first found that the fluorescence of the “negative” sample was due to the primer used in PCR. Next, it was found that nonspecific fluorescence generation by this primer can be suppressed by adjusting the pH of the reaction solution after PCR. That is, if the pH of the reaction solution is kept at 10-12,
Fluorescence from the primers in the "negative" sample was completely imperceptible by visual observation. On the other hand, DNA that was present in the "positive" sample and amplified by PCR under this pH condition.
Can form a double-stranded chain, and thus reacts specifically with a fluorescent substance to emit fluorescence, making it possible to reliably distinguish a “negative” sample from a “positive” sample by visual observation. It was
【００１１】これらが相俟って、本発明者等は従来技術
における既述の課題を解決し、本発明を完成するに至っ
た。Together with these, the present inventors have completed the present invention by solving the above-mentioned problems in the prior art.
【００１２】[0012]
【実施例】次に、実施例及び試験例により本発明を更に
詳細に且つ具体的に説明する。尚、以下の実施例及び試
験例においては蛍光物質として臭化エチジウムが用いら
れたが、DNA に親和性を有するものであれば、他の蛍光
物質も同様に用い得ることに留意され度い。EXAMPLES Next, the present invention will be described in more detail and specifically with reference to Examples and Test Examples. Although ethidium bromide was used as the fluorescent substance in the following examples and test examples, it should be noted that other fluorescent substances can also be used as long as they have an affinity for DNA.
【００１３】実施例 PCR によるヒト C 型肝炎ウイルス遺伝子 DNA の検出に
ついて説明する。本発明者等が開発した方法 (特願平 3
- 27537 明細書) に従い、ヒト C 型肝炎ウイルスに特
異的な2 組のプライマーを用い且つ C 型肝炎と確定診
断された患者 10 名の血清と健常人 10 名の血清をそれ
ぞれ対象検体とし、連続して 2回の PCR を行った。こ
の PCR 後の反応液をポリアクリルアミドゲル電気泳動
により確認した処、C 型肝炎患者の血清全例において、
予測された 180 塩基対の位置にバンドが検出され、こ
れによって PCR 産物である DNA の存在が確認された。
一方、健常人の血清を検体としたものに関しては、何れ
の場合にも当該バンドは検出されなかった。従って、上
記の 20 検体は検出試験に供するのに適切な検体である
ことが判明したので、これらの検体を用いて本発明によ
る検出法を下記のようにして実施した。[0013] will be described the detection of human hepatitis C virus gene DNA according to the embodiment PCR. Method developed by the present inventors (Japanese Patent Application No.
-27537 specification), using two sets of primers specific for human hepatitis C virus, and sera of 10 patients with a definite diagnosis of hepatitis C and sera of 10 healthy individuals were used as target samples, and Then, PCR was performed twice. When the reaction solution after this PCR was confirmed by polyacrylamide gel electrophoresis, all the sera from patients with hepatitis C were
A band was detected at the predicted position of 180 base pairs, confirming the presence of the PCR product, DNA.
On the other hand, the band was not detected in any of the cases in which the serum of a healthy person was used as the sample. Therefore, the above 20 samples were found to be suitable for the detection test, and the detection method according to the present invention was carried out as follows using these samples.
【００１４】各検体に関して上記のように 2 回の PCR
を行い、PCR 後の反応チューブに直接、1M グリシン-Na
OH 緩衝液 (pH 12) を 20μl と、10μg/ml の臭化エチ
ジウム溶液を 8μl 添加することにより pHを 11.1 に
調整した (上記の操作を、本明細書においては「pH 調
整」と称する)。この pH 調整された反応液を収容して
いる反応チューブに、トランスイルミネーター或は UV
ランプによる UV 照射を暗所において行い蛍光の有無を
目視観察した。結果は図１に示される通りであり、C 型
肝炎患者由来の血清検体全例で明瞭な蛍光が認められ、
健常人由来の血清検体では蛍光は全く認められなかっ
た。Two PCRs as above for each sample
Perform 1M glycine-Na directly in the reaction tube after PCR.
The pH was adjusted to 11.1 by adding 20 μl of OH buffer (pH 12) and 8 μl of 10 μg / ml ethidium bromide solution (the above operation is referred to as “pH adjustment” in the present specification). Insert a transilluminator or UV into the reaction tube containing the pH-adjusted reaction solution.
UV irradiation with a lamp was performed in the dark and the presence or absence of fluorescence was visually observed. The results are shown in Fig. 1, and clear fluorescence was observed in all the serum samples derived from hepatitis C patients.
No fluorescence was observed in the serum samples from healthy individuals.
【００１５】各反応液が発している蛍光の強度を確認す
るために、反応チューブから反応液を取り出し、蛍光強
度計を用い励起波長 312nm、検出波長 590nm にて蛍光
強度を測定した。結果は図２に示される通りであり。健
常人由来の血清を検体とした場合の反応液の蛍光強度は
全例で低く、一方 C 型肝炎患者由来の血清を検体とし
た場合の反応液の蛍光強度値は健常人由来の血清を検体
とした場合の反応液の蛍光強度値の約 5 倍であり、目
視観察で確認された蛍光の強さと一致していた。このこ
とは、本発明による検出法が目視観察による判定を可能
にするものであり、その判定結果は十分信頼できるもの
であることを示している。In order to confirm the intensity of fluorescence emitted from each reaction solution, the reaction solution was taken out from the reaction tube and the fluorescence intensity was measured with a fluorescence intensity meter at an excitation wavelength of 312 nm and a detection wavelength of 590 nm. The results are as shown in Figure 2. The fluorescence intensity of the reaction solution when using serum from a healthy person as a sample is low in all cases, while the fluorescence intensity value of the reaction solution when using serum from a patient with hepatitis C as a sample The fluorescence intensity value of the reaction solution was about 5 times, which was in agreement with the fluorescence intensity confirmed by visual observation. This indicates that the detection method according to the present invention enables judgment by visual observation, and the judgment result is sufficiently reliable.
【００１６】試験例 PCR 後の反応チューブに臭化エチジウムを添加したが、
pH 調整を行わなかったもの (対照品) と、本発明方法
に従い臭化エチジウムを添加し且つ pH 調整を施したも
の (被験品) とについて、上記の実施例と同様に蛍光強
度を測定して比較した。結果は下記の表１に示される通
りであり (表１中において、値はそれぞれ 5例の平均値
で示されている)、この結果から pH 調整を行わない場
合には、健常人由来の血清検体に関する反応液の蛍光強
度は約 10 であって可成り高く、C 型肝炎患者由来の血
清検体に関する反応液の蛍光強度との差が少ないので、
「陰性」か「陽性」かの目視観察による判定が難しい状
況にあること、本発明方法に従って pH 調整を行えば、
健常人由来の血清検体に関する反応液の蛍光強度は約 1
/4 になり、目視観察では蛍光が殆ど感知できなくなる
こと、並びに C 型肝炎患者由来の血清検体に関する反
応液の蛍光強度も低下するが、低下の程度は約 1/2.5
であり、C 型肝炎患者由来の血清検体に関する反応液の
蛍光強度との差が約5 倍程度あり、蛍光を目視観察で充
分に確認することができ、従って「陰性」検体と「陽
性」検体との判定を容易に下し得ることが判る。 Test Example Ethidium bromide was added to the reaction tube after PCR,
Fluorescence intensity was measured in the same manner as in the above-mentioned Examples for those without pH adjustment (control product) and those for which ethidium bromide was added according to the method of the present invention and pH adjustment was performed (test product). Compared. The results are as shown in Table 1 below (in Table 1, the values are shown as the average value of 5 cases). From these results, when pH adjustment was not performed, the serum derived from a healthy person was obtained. The fluorescence intensity of the reaction solution for the sample is about 10, which is quite high, and there is little difference from the fluorescence intensity of the reaction solution for the serum sample derived from a hepatitis C patient.
When it is difficult to determine by "visual observation" whether "negative" or "positive", if the pH is adjusted according to the method of the present invention,
The fluorescence intensity of the reaction solution for a serum sample from a healthy person is about 1
The fluorescence intensity becomes almost undetectable by visual observation, and the fluorescence intensity of the reaction solution for serum samples from hepatitis C patients also decreases, but the extent of the decrease is about 1 / 2.5.
The difference between the fluorescence intensity of the reaction solution and that of the serum sample derived from hepatitis C patient is about 5 times, and the fluorescence can be sufficiently confirmed by visual observation. Therefore, the “negative” sample and the “positive” sample can be confirmed. It turns out that the judgment can be easily made.
【００１７】[0017]
【表１】 [Table 1]
【００１８】[0018]
【発明の効果】本発明による DNA の検出法は、PCR に
より増幅した反応チューブ内の DNA と蛍光物質の結合
による蛍光発色を目視観察によっても確認できる方法な
ので、従来の PCR 産物の電気泳動、臭化エチジウム染
色、ＵＶ照射による反応産物の確認及び写真撮影等の一
連の操作の内で、PCR 産物の電気泳動や写真撮影と云う
煩雑な操作が要求されず、従って極めて簡便であり且つ
所要時間が大幅に短縮する。更に、PCR 産物を反応チュ
ーブから取り出す必要もないので、PCR の欠点とされる
コンタミネーションの問題も生じない。従って、本発明
による DNA の検出法によれば単位時間当りにおいて多
数の検体を処理することができ、しかも簡便であるため
に目視観察による判定を機器による判定に置き換えるこ
とにより検出の自動化ないし半自動化を可能にする。EFFECTS OF THE INVENTION The method for detecting DNA according to the present invention is a method in which the fluorescent color generated by the binding of the fluorescent substance to the DNA in the reaction tube amplified by PCR can be confirmed by visual observation. In a series of operations such as ethidium bromide staining, confirmation of reaction products by UV irradiation, and photography, complicated operations such as electrophoresis and photography of PCR products are not required, and therefore, it is extremely simple and time required. Significantly shorten. Furthermore, since it is not necessary to remove the PCR product from the reaction tube, the problem of contamination, which is a drawback of PCR, does not occur. Therefore, according to the method for detecting DNA according to the present invention, a large number of samples can be processed per unit time, and since it is simple, the determination by visual observation is replaced with the determination by an instrument, so that the detection is automated or semi-automated. To enable.
【図１】C 型肝炎と確定診断された患者 10 名由来の血
清と、健常人 10 名由来の血清を検体とし且つ C 型肝
炎ウイルス遺伝子 DNA に特異的な 2 組のプライマーを
用いて反応チューブ内で PCR を 2 回実施し、この PCR
後の反応液に蛍光物質である臭化エチジウムと pH 調
整のための緩衝液を添加し、暗所にて反応チューブにUV
照射を行って蛍光の有無、即ち PCR により増幅した D
NA の存否を目視観察により判定し、その確認のために
写真撮影した撮像を模写した図である。[Fig. 1] Reaction tubes using sera derived from 10 patients with a definite diagnosis of hepatitis C and sera derived from 10 healthy persons, and using two sets of primers specific for hepatitis C virus gene DNA. PCR was performed twice in the
Add ethidium bromide, which is a fluorescent substance, and a buffer solution for pH adjustment to the subsequent reaction solution, and add UV to the reaction tube in the dark.
Existence of fluorescence by irradiation, that is, D amplified by PCR
It is the figure which imitated the presence or absence of NA by visual observation and imitated the photography image-taking for the confirmation.
【図２】図１に示された蛍光の強度を、蛍光強度計を用
い励起波長 312nm、検出波長 590nm にて測定した結果
を示すグラフである。FIG. 2 is a graph showing the results of measuring the fluorescence intensity shown in FIG. 1 using a fluorescence intensity meter at an excitation wavelength of 312 nm and a detection wavelength of 590 nm.
フロントページの続き (72)発明者 城森 孝仁 愛知県名古屋市東区東外堀町35番地 株式 会社三和化学研究所内 (72)発明者 高橋 治雄 愛知県名古屋市東区東外堀町35番地 株式 会社三和化学研究所内 (72)発明者 林 祐二 愛知県名古屋市東区東外堀町35番地 株式 会社三和化学研究所内 (72)発明者 鈴木 栄二 愛知県名古屋市東区東外堀町35番地 株式 会社三和化学研究所内Front page continued (72) Inventor Takahito Shiromori 35 Higashi Sotobori-cho, Higashi-ku, Aichi Prefecture Sanwa Chemical Research Institute (72) Inventor Haruo Takahashi 35 Higashi-Tobori-cho, Higashi-ku Nagoya City, Aichi Sanwa Co., Ltd. Inside the Chemical Research Laboratory (72) Inventor Yuji Hayashi 35, Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Prefecture Sanwa Chemical Research Institute (72) Inventor Eiji Suzuki 35, Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Prefecture Sanwa Chemical Research Company In-house
Claims (5)
応チューブ内で増幅した産物である DNA を、該 DNA に
親和性を有する蛍光物質と結合させて検出する際に、PC
R 後に反応液の pH を調整して非特異的な蛍光の発生を
抑制することを特徴とする、ポリメラーゼ連鎖反応によ
り増幅した DNA の検出法。1. When a DNA, which is a product amplified in a reaction tube by polymerase chain reaction (PCR), is bound to a fluorescent substance having an affinity for the DNA for detection, a PC is used.
A method for detecting DNA amplified by polymerase chain reaction, which comprises controlling the pH of the reaction solution after R to suppress the generation of non-specific fluorescence.
整することを特徴とする、請求項１に記載のポリメラー
ゼ連鎖反応により増幅した DNA の検出法。2. The method for detecting DNA amplified by the polymerase chain reaction according to claim 1, wherein the pH of the reaction solution is adjusted to 10 −12 after PCR.
出さず、反応チューブに特定波長の光を照射し、蛍光の
有無を目視観察することを特徴とする、請求項１又は２
に記載のポリメラーゼ連鎖反応により増幅した DNA の
検出法。3. The reaction solution after PCR is not taken out from the reaction tube, but the reaction tube is irradiated with light having a specific wavelength, and the presence or absence of fluorescence is visually observed.
A method for detecting DNA amplified by the polymerase chain reaction according to 1.
物、ウイルス、プラスミド及び遺伝子組換え体由来の D
NA 若しくは RNAを鋳型としたものであることを特徴と
する、請求項１、２又は３に記載のポリメラーゼ連鎖反
応により増幅した DNA の検出法。4. The product of DNA amplified by PCR is D derived from organisms, viruses, plasmids and recombinants.
The method for detecting DNA amplified by the polymerase chain reaction according to claim 1, characterized in that NA or RNA is used as a template.
質、抗癌剤、DNA 及びRNA の標識用蛍光試薬並びに核染
色用蛍光色素から選ばれたものであることを特徴とする
請求項１、２、３又は４に記載のポリメラーゼ連鎖反応
により増幅した DNA の検出法。5. The fluorescent substance having an affinity for DNA is selected from antibiotics, anticancer agents, fluorescent reagents for labeling DNA and RNA, and fluorescent dyes for nuclear staining. A method for detecting DNA amplified by the polymerase chain reaction according to 3 or 4.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19987991A JPH0568596A (en) | 1991-07-16 | 1991-07-16 | Detection of dna multiplied by polymerase chain reaction |
| EP91120311A EP0488243A1 (en) | 1990-11-30 | 1991-11-27 | Method of extracting virus genome from sample derived from living body infected with the virus and detecting the genome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19987991A JPH0568596A (en) | 1991-07-16 | 1991-07-16 | Detection of dna multiplied by polymerase chain reaction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0568596A true JPH0568596A (en) | 1993-03-23 |
Family
ID=16415137
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP19987991A Pending JPH0568596A (en) | 1990-11-30 | 1991-07-16 | Detection of dna multiplied by polymerase chain reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0568596A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63102677A (en) * | 1986-08-22 | 1988-05-07 | エフ．ホフマン―ラ ロシュ アクチェンゲゼルシャフト | Heat stable enzyme and its use |
-
1991
- 1991-07-16 JP JP19987991A patent/JPH0568596A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63102677A (en) * | 1986-08-22 | 1988-05-07 | エフ．ホフマン―ラ ロシュ アクチェンゲゼルシャフト | Heat stable enzyme and its use |
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