JP4854334B2 - 核酸増幅生成物のリアルタイム検出装置 - Google Patents
核酸増幅生成物のリアルタイム検出装置 Download PDFInfo
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- JP4854334B2 JP4854334B2 JP2006059381A JP2006059381A JP4854334B2 JP 4854334 B2 JP4854334 B2 JP 4854334B2 JP 2006059381 A JP2006059381 A JP 2006059381A JP 2006059381 A JP2006059381 A JP 2006059381A JP 4854334 B2 JP4854334 B2 JP 4854334B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
[DNA]=[DNA]0(1+e)c ・・・・(1)
[DNA] :PCRプロダクトの濃度
[DNA]0:標的テンプレートの初期濃度
e:平均PCR効率
c:サイクル数
・dNTPとプライマーの加水分解
・DNAポリメラーゼ(テンプレート(鋳型)のコピーをつくるためのDNA合成酵素)の熱による失活
・1本鎖PCRフラグメントの再会合によるプライマーアニーリング効率の低下
・非特異的PCRプロダクトによる競合素材
・ピロホスフェートのようなPCR阻害物質の蓄積
・DNAポリメラーゼのエキソヌクレアーゼ活性によるPCRプロダクトの加水分解
・光学系の誤差
・校正溶液の濃度誤差
・校正溶液の分注誤差
・反応容器の蓋やシールフィルムの光透過性誤差
・反応容器の汚れ誤差
・反応溶液の分注誤差
前述した如く反応開始前において、反応検出装置1のカメラ27で撮影された画像データでは、各ピクセルの輝度は必ずしも一定では無く、図4の(1)に示すように光学系の汚れや迷走光などにより斑が発生する。反応が進むとこの斑は図5の(2)に示すようにウエル7Aの画像に重なってウエル7A本来の蛍光強度に冗長されてしまう。
[DNA]realB11=[DNA]rawB11−(([DNA]bg1+[DNA]bg2+[DNA]bg3+[DNA]bg4)/4) ・・・・(2)
次に、図8は96個の全てのウエル7Aの増幅曲線を示している。横軸は温度サイクル数、縦軸は蛍光強度である。前述した如く図5、図6の上半分のXグループと下半分のYグループで濃度の異なる反応溶液が分注してある関係上、本来ならば反応曲線は2本の線に見えなければならない。また、閾値サイクル数Ctも2点となるはずである。しかしながら、前述した光学系の誤差、校正溶液の濃度誤差、校正溶液の分注誤差や反応溶液の分注誤差などの装置上の誤差要因により、各ウエル7Aの増幅曲線はバラツキ、閾値サイクル数Ctも(8)、(9)のように複数生じる(バラツキ)。
[DNA]nN=[DNA]n/([DNA]max+Z) ・・・(3)
ここで、サイクル後期のプラトー領域で蛍光強度がばらつく要因としては、上述した装置上の誤差要因だけでなく、ケミカルな反応のバラツキによっても発生する。そして、その要因に関しては指数関数的増幅領域の蛍光強度に比例的に影響しないことが考えられる。即ち、プラトー効果の領域での蛍光強度を完全に一致させないほうが良い場合もある。
Z=α[DNA]max+β ・・・(4)
α、βは各ウエル7Aに共通する係数。
また、演算処理部31は前述した最大値[DNA]maxを、蛍光強度の移動平均で算出している。なぜならば蛍光強度[DNA]realは実際にはのこぎり状を呈するからである。しかしながら、正規化に使用する最大値としては、このノコギリのピーク値であってもよく、当該ピーク値の例えば90%などの数値(何れも最大値に関連する値)を使用しても差し支えない。
ここで、図8の(10)はサイクル初期で反応結果を検出できないレベルの領域であるが、ウエル7A内の反応溶液そのものが最初から一定の蛍光を発生しているので、この領域でも一般的には蛍光強度は0では無い。そこで、キーボード(マウス)33からの指示に基づき、演算処理部31はベースラインの補正を行う。即ち、演算処理部31は、サイクル初期の領域(10)での各ウエル7Aの蛍光強度の平均値[DNA]baseをベースラインとし、この平均値を前述した[DNA]n及び[DNA]maxから差し引いてから上述した正規化の処理を行っている。図9、図10で初期の蛍光強度が0となっているのは、この処理を行っているためである([DNA]baseは[DNA]nの最小値を使用しても差し支えない)。
7 反応容器
7A ウエル(反応領域)
10 ペルチェ素子
27 カメラ
31 演算処理部
34 ディスプレイ
C 処理装置
R リアルタイム検出装置
Claims (1)
- ウエル又はチューブにて構成された複数の反応領域に温度サイクルを与え、各反応領域における核酸増幅生成物からの蛍光強度をリアルタイムで検出する装置であって、
前記反応領域から得られる蛍光測定値[DNA]rawを検出し、該蛍光測定値[DNA]rawの検出の都度、前記反応領域近傍における複数の反応領域外領域から得られる蛍光測定値の単純平均、若しくは、所定の重み付けを行った後の平均値としての蛍光測定値[DNA]bgを算出し、前記蛍光測定値[DNA]rawから前記蛍光測定値[DNA]bgを差し引くことにより、当該反応領域の蛍光強度[DNA]realを決定することを特徴とする核酸増幅生成物のリアルタイム検出装置。
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006059381A JP4854334B2 (ja) | 2006-03-06 | 2006-03-06 | 核酸増幅生成物のリアルタイム検出装置 |
CN201310077508.7A CN103232936B (zh) | 2006-03-06 | 2007-03-01 | 核酸扩增产物实时检测装置 |
US12/281,540 US20090155891A1 (en) | 2006-03-06 | 2007-03-01 | Apparatus for detecting nucleic acid amplification product in real time |
EP07737629.1A EP1992682B1 (en) | 2006-03-06 | 2007-03-01 | Apparatus for real-time detection of nucleic acid amplification product |
PCT/JP2007/053949 WO2007102400A1 (ja) | 2006-03-06 | 2007-03-01 | 核酸増幅生成物のリアルタイム検出装置 |
CN2007800080432A CN101395261B (zh) | 2006-03-06 | 2007-03-01 | 核酸扩增产物实时检测装置 |
KR1020087021716A KR101344673B1 (ko) | 2006-03-06 | 2008-09-05 | 핵산 증폭 생성물의 실시간 검출 장치 |
US14/339,092 US10544453B2 (en) | 2006-03-06 | 2014-07-23 | Method for detecting nucleic acid amplification product in real time |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006059381A JP4854334B2 (ja) | 2006-03-06 | 2006-03-06 | 核酸増幅生成物のリアルタイム検出装置 |
Publications (2)
Publication Number | Publication Date |
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JP2007236219A JP2007236219A (ja) | 2007-09-20 |
JP4854334B2 true JP4854334B2 (ja) | 2012-01-18 |
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JP2006059381A Active JP4854334B2 (ja) | 2006-03-06 | 2006-03-06 | 核酸増幅生成物のリアルタイム検出装置 |
Country Status (6)
Country | Link |
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US (2) | US20090155891A1 (ja) |
EP (1) | EP1992682B1 (ja) |
JP (1) | JP4854334B2 (ja) |
KR (1) | KR101344673B1 (ja) |
CN (2) | CN103232936B (ja) |
WO (1) | WO2007102400A1 (ja) |
Cited By (1)
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US11530989B2 (en) | 2019-07-26 | 2022-12-20 | Fujifilm Corporation | Fluorescence imaging device |
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JP5148387B2 (ja) | 2008-06-30 | 2013-02-20 | 浜松ホトニクス株式会社 | 分光測定装置、分光測定方法、及び分光測定プログラム |
JP5339838B2 (ja) * | 2008-10-01 | 2013-11-13 | キヤノン株式会社 | 遺伝子検査装置 |
JP2010197251A (ja) * | 2009-02-25 | 2010-09-09 | Fujitsu Ltd | 撮影装置、撮影方法および撮影プログラム |
WO2011021640A1 (ja) * | 2009-08-20 | 2011-02-24 | タカラバイオ株式会社 | 温度サイクル装置 |
JP5744038B2 (ja) * | 2009-10-21 | 2015-07-01 | コーニンクレッカ フィリップス エヌ ヴェ | 増幅反応の解析ツール |
US20110276317A1 (en) * | 2010-04-11 | 2011-11-10 | Life Technologies Corporation | SYSTEMS AND METHODS FOR MODEL-BASED qPCR |
JP2011250714A (ja) * | 2010-05-31 | 2011-12-15 | Sanyo Electric Co Ltd | 増幅装置、検出装置 |
EP2959284A2 (en) * | 2013-02-22 | 2015-12-30 | Life Technologies Corporation | Optical systems and methods for biological analysis |
WO2015054259A1 (en) * | 2013-10-07 | 2015-04-16 | Rutgers, The State University Of New Jersey | Systems and methods for determining an unknown characteristic of a sample |
KR101670232B1 (ko) | 2013-10-28 | 2016-10-31 | 한국과학기술연구원 | 다공성 구조체 및 이의 제조방법 |
KR102078085B1 (ko) | 2013-12-31 | 2020-02-17 | 주식회사 미코바이오메드 | 농식품의 식중독균 검출용 랩온어칩 기반의 초고속 실시간 pcr 장치, 및 이를 이용한 식중독 검출방법 |
ES2899739T3 (es) * | 2014-08-27 | 2022-03-14 | Hoffmann La Roche | Un procedimiento de análisis y sistema para analizar una reacción de amplificación de ácido nucleico |
KR101751700B1 (ko) | 2014-09-18 | 2017-06-30 | 한국과학기술연구원 | 전하를 띄는 다공성 구조체 |
WO2016127124A2 (en) * | 2015-02-06 | 2016-08-11 | Life Technologies Corporation | Methods and systems for biological instrument calibration |
KR101872803B1 (ko) | 2016-05-03 | 2018-06-29 | 한국과학기술연구원 | 핵산 프라이머-탄소재료 복합체를 포함하는 다공성 구조체 및 이를 이용한 핵산 증폭 방법 |
CN107083426A (zh) * | 2017-03-30 | 2017-08-22 | 杭州晶格科学仪器有限公司 | 一种荧光定量检测方法 |
CN107746806B (zh) * | 2017-11-20 | 2024-02-20 | 鲲鹏基因(北京)科技有限责任公司 | 一种实时荧光定量pcr仪 |
JP7221491B2 (ja) * | 2018-09-18 | 2023-02-14 | 株式会社ミズホメディー | 複数の標的核酸を検出するキットを用いる検出方法 |
JP2021153516A (ja) * | 2020-03-27 | 2021-10-07 | シスメックス株式会社 | 核酸増幅の成否判定方法、核酸増幅の成否判定装置、及び核酸増幅の成否判定システム |
FR3136092A1 (fr) * | 2022-05-27 | 2023-12-01 | Bforcure | Dispositif de traitement de mesures de PCR temps réel à milieu de réaction homogène dans lequel les réactifs de PCR peuvent diffuser |
CN115197835B (zh) * | 2022-09-15 | 2023-07-11 | 浙江正合谷生物科技有限公司 | 一种基于自学习的核酸扩增荧光定量温控系统 |
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- 2006-03-06 JP JP2006059381A patent/JP4854334B2/ja active Active
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2007
- 2007-03-01 CN CN201310077508.7A patent/CN103232936B/zh active Active
- 2007-03-01 US US12/281,540 patent/US20090155891A1/en not_active Abandoned
- 2007-03-01 EP EP07737629.1A patent/EP1992682B1/en active Active
- 2007-03-01 WO PCT/JP2007/053949 patent/WO2007102400A1/ja active Application Filing
- 2007-03-01 CN CN2007800080432A patent/CN101395261B/zh active Active
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- 2014-07-23 US US14/339,092 patent/US10544453B2/en active Active
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US11530989B2 (en) | 2019-07-26 | 2022-12-20 | Fujifilm Corporation | Fluorescence imaging device |
Also Published As
Publication number | Publication date |
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CN101395261B (zh) | 2013-11-06 |
US20140335531A1 (en) | 2014-11-13 |
EP1992682A4 (en) | 2010-12-22 |
JP2007236219A (ja) | 2007-09-20 |
CN103232936B (zh) | 2016-04-06 |
KR101344673B1 (ko) | 2013-12-23 |
US10544453B2 (en) | 2020-01-28 |
WO2007102400A1 (ja) | 2007-09-13 |
CN101395261A (zh) | 2009-03-25 |
US20090155891A1 (en) | 2009-06-18 |
CN103232936A (zh) | 2013-08-07 |
EP1992682B1 (en) | 2014-07-23 |
EP1992682A1 (en) | 2008-11-19 |
KR20080103548A (ko) | 2008-11-27 |
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