JP7223689B2 - 反応処理装置 - Google Patents
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Description
また、流路12は第2フィルタ直前で2系統になっている。蒸発した試料の一部が液滴となって流路内部に付着する場合があり、特に比較的高温に維持されることが予定されている第2フィルタ30付近では付着した液滴が試料の液送を妨げる虞があり、それを補償するために2系統となっている。
また、第1空気連通口24、第2空気連通口26も基板14の下面14aに露出しているが、これらを封止するために図1(c)で示したように流路封止フィルム16とは別体の第1封止フィルム18を用いた。
さらに、第1試料導入口45、第2試料導入口46、接続流路241および261を封止するために図1(a)で示したように第2封止フィルム20が基板14の上面14bに貼り付けられる。流路封止フィルム16に加え、第1封止フィルム18および第2封止フィルム20を貼った状態では、全流路は閉空間となっている。
第2封止フィルム20には、剥がすときに手指で持ちやすく容易に剥がす作業をできるように粘着性のないタブ201が形成されていてもよい。作業者はタブ201を持って、例えば図1(a)で示した点線(A)の所まで第2封止フィルム20を剥がす。
(ii)上記(i)で増幅される領域の鋳型DNAを合成DNA製造メーカーに依頼し1×106Copies/μL濃度で作製。これから10倍の希釈列からなる標準検体(1×101~1×106Copies/μL)を作製した。
(iii)さらに、試薬には、DNAポリメラーゼとしてタカラバイオ株式会社製SpeedSTAR(登録商標)HS DNA Polymeraseを0.1U/μLと、これに付属の×10Fast buffer IとdNTP Mixとを説明書通りに混合して添加した。なお、[U/μL]の「U」=ユニットは、酵素活性量の単位であり、至適条件で酵素が1分間に1μmol(1×10-6mol)の基質を変換することができる酵素の量が1Uと定義され、大まかには酵素の量を表す。
(iv)この試薬24μLに、(ii)で作成した濃度の異なる標準検体1μLを添加し試料とした。さらに、ネガティブコントロール(ネガコン)の試料として標準検体を添加しない試薬を準備した。なお、ネガティブコントロールとは、効果の対照実験で陰性と結果されるべき対照(試料)であり、この場合はPCRを行っても、菌は含まれない(陰性)という結果をもたらすものである。この反対はポジティブコントロール(ポジコン)であり、上記の標準検体が該当し、PCRを行うことにより菌が含まれる(陽性)という結果をもたらすものである。
配列番号2:リバースPCRプライマー
配列番号3:プローブ
Claims (7)
- 試料が移動する流路と、前記流路の両端に設けられた一対の空気連通口と、前記流路における前記一対の空気連通口の間に設けられた第1温度領域および第2温度領域とを備える反応処理容器であって、前記第1温度領域および前記第2温度領域がそれぞれ曲線部と直線部とを組み合わせた蛇行状流路を含む、反応処理容器と、
前記第1温度領域を第1温度に維持するとともに、前記第2温度領域を前記第1温度よりも高い第2温度に維持する温度制御手段と、
試料を前記流路における前記第1温度領域と前記第2温度領域との間で往復式に移動させるために空気の吐出および吸引を行う送液システムと、
を備え、
前記反応処理容器の前記一対の空気連通口のうち前記第2温度領域から遠い方の空気連通口は、チューブを介して前記送液システムに連通され、
前記反応処理容器の前記一対の空気連通口のうち前記第2温度領域に近い方の空気連通口は、大気圧に開放されることを特徴とする反応処理装置。 - 前記送液システムは、
一定の体積を有する内部空間と、内部空間と外部とを連通する第1空気口および第2空気口とを有するチャンバと、
前記チャンバの内部空間に前記第1空気口から空気を吐出するよう配置された第1ポンプと、
前記チャンバの内部空間に前記第2空気口から空気を吐出するよう配置された第2ポンプと、を備え、
前記チャンバの第1空気口は、前記チューブを介して前記反応処理容器の前記一対の空気連通口のうち前記第2温度領域から遠い方の空気連通口に連通され、
前記チャンバの第2空気口は、大気圧に開放され、
前記第1ポンプと前記第2ポンプは、交互に空気の吐出動作を行うよう制御されることを特徴とする請求項1に記載の反応処理装置。 - 前記反応処理容器の前記流路の両端のうち少なくとも前記第2温度領域から遠い方の流路端部に、フィルタが設けられることを特徴とする請求項1または2に記載の反応処理装置。
- 前記反応処理容器は、前記第1温度領域と前記第2温度領域とを接続する接続流路を備え、
前記接続流路を通過する試料からの蛍光を検出する蛍光検出器をさらに備えることを特徴とする請求項1から3のいずれかに記載の反応処理装置。 - 前記反応処理容器における前記流路は、前記第1温度よりも低い第3温度に維持される第3温度領域をさらに備え、
前記第3温度領域は、前記第1温度領域と、前記一対の空気連通口のうち前記第2温度領域から遠い方の空気連通口との間に設けられ、
前記反応処理容器は、前記第3温度領域に試料を導入するための試料導入口を備えることを特徴とする請求項1から4のいずれかに記載の反応処理装置。 - 前記第1温度領域と前記第2温度領域との間隔は、前記第1温度領域および前記第2温度領域に含まれる前記蛇行状流路を構成する前記直線部の長さより長いことを特徴とする請求項1から5のいずれかに記載の反応処理装置。
- 前記反応処理容器は、前記第1温度領域と前記第2温度領域とを接続する接続流路を備え、
前記接続流路の長さは、前記第1温度領域および前記第2温度領域に含まれる前記蛇行状流路を構成する前記直線部の長さより長いことを特徴とする請求項1から6のいずれかに記載の反応処理装置。
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PCT/JP2018/023073 WO2018235766A1 (ja) | 2017-06-23 | 2018-06-18 | 反応処理装置 |
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US (1) | US11529631B2 (ja) |
EP (1) | EP3643778A4 (ja) |
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WO2019139135A1 (ja) * | 2018-01-15 | 2019-07-18 | 日本板硝子株式会社 | 反応処理装置 |
EP3940053A4 (en) * | 2019-03-15 | 2023-01-04 | National Institute Of Advanced Industrial Science And Technology | NUCLEIC ACID AMPLIFICATION PROCESS |
JP6732994B1 (ja) * | 2019-04-05 | 2020-07-29 | 日本板硝子株式会社 | 反応処理容器 |
JP1653576S (ja) * | 2019-04-09 | 2020-02-25 | ||
USD928344S1 (en) * | 2019-04-23 | 2021-08-17 | Nippon Sheet Glass Company, Limited | Gene amplification chip for medical and laboratory use |
JP6652673B1 (ja) * | 2019-06-07 | 2020-02-26 | 日本板硝子株式会社 | 反応処理容器 |
US11465143B2 (en) | 2019-06-07 | 2022-10-11 | Nippon Sheet Glass Company, Limited | Reaction processing vessel |
JP2020198867A (ja) * | 2020-01-23 | 2020-12-17 | 日本板硝子株式会社 | 反応処理容器 |
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JP2009232700A (ja) | 2008-03-26 | 2009-10-15 | Shimadzu Corp | 反応処理方法及び反応処理装置 |
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WO2017094674A1 (ja) | 2015-12-01 | 2017-06-08 | 日本板硝子株式会社 | Pcr反応容器、pcr装置およびpcr方法 |
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US20100167384A1 (en) | 2005-11-30 | 2010-07-01 | Micronics, Inc, | Microfluidic mixing and analytical apparatus |
JP2009232700A (ja) | 2008-03-26 | 2009-10-15 | Shimadzu Corp | 反応処理方法及び反応処理装置 |
WO2017094674A1 (ja) | 2015-12-01 | 2017-06-08 | 日本板硝子株式会社 | Pcr反応容器、pcr装置およびpcr方法 |
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