JP6117775B2 - スタフィロコッカス・アウレウスの検出のための組成物及び方法 - Google Patents
スタフィロコッカス・アウレウスの検出のための組成物及び方法 Download PDFInfo
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Description
本発明は微生物の診断分野に関し、より具体的にはスタフィロコッカス・アウレウス(Staphylococcus aureus)の検出に関する。
莢膜多糖酵素遺伝子標的の選択
CPEオリゴの性能評価法
排他性評価法
Claims (10)
- − 配列番号2及び17〜20からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第一のオリゴヌクレオチド;並びに
− 配列番号3、6、25及び26からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第二のオリゴヌクレオチド;
を含むか、或いは、
− 配列番号8、12及び14〜16からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第一のオリゴヌクレオチド;並びに
− 配列番号9及び21〜24からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第二のオリゴヌクレオチド;
を含む、スタフィロコッカス・アウレウス(Staphylococcus aureus)(SA)を検出するためのオリゴヌクレオチドのセット。 - 40個以下のヌクレオチドを有する、検出可能に標識された第三のオリゴヌクレオチドをさらに含み、ここで前記第三のオリゴヌクレオチドは、
前記第一のオリゴヌクレオチドが、配列番号2及び17〜20からなる群から選択される配列又はその相補配列を含み、前記第二のオリゴヌクレオチドが、配列番号3、6、25及び26からなる群から選択される配列又はその相補配列を含む場合には、配列番号4、27〜29及び32〜34からなる群から選択される配列又はその相補配列を含み、或いは、
前記第一のオリゴヌクレオチドが、配列番号8、12及び14〜16からなる群から選択される配列又はその相補配列を含み、前記第二のオリゴヌクレオチドが、配列番号9及び21〜24からなる群から選択される配列又はその相補配列を含む場合には、配列番号10、30及び31からなる群から選択される配列又はその相補配列を含む、
請求項1に記載のオリゴヌクレオチドのセット。 - 試料中のスタフィロコッカス・アウレウス(Staphylococcus aureus)(SA)を検出する方法であって:
− 前記試料を1組のSAプライマーと接触させて、SAが前記試料中に存在する場合には増幅産物を生産することを含む、増幅ステップを行い;
− 前記増幅産物を、1又は2以上の検出可能なSAプローブと接触させることを含む、ハイブリダイズステップを行い;そして
− 前記増幅産物の存在又は不存在を検出すること、ここで、前記増幅産物の存在は前記試料中のSAの存在の指標となり、そして前記増幅産物の不存在は前記試料中のSAの不存在の指標となる、
を含み、
ここで、前記1組のSAプライマーは、配列番号2及び17〜20からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第一のオリゴヌクレオチドプライマー、並びに、配列番号3、6、25及び26からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第二のオリゴヌクレオチドプライマーを含み、そして前記1又は2以上の検出可能なSAプローブは、配列番号4、27〜29及び32〜34からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有するか、或いは、
前記1組のSAプライマーは、配列番号8、12及び14〜16からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第一のオリゴヌクレオチドプライマー、並びに、配列番号9及び21〜24からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第二のオリゴヌクレオチドプライマーを含み、そして前記1又は2以上の検出可能なSAプローブは、配列番号10、30及び31からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、前記方法。 - − 前記ハイブリダイズステップは、前記増幅産物を、ドナー蛍光部位及び対応するアクセプター蛍光部位で標識されるプローブと接触させることを含み;そして
− 前記検出ステップは、前記プローブのドナー蛍光部位とアクセプター蛍光部位との間の蛍光共鳴エネルギー移動(FRET)の存在又は不存在を検出することを含み、ここで蛍光の存在又は不存在は、前記試料中のSAの存在又は不存在の指標となる、請求項3に記載の方法。 - 前記増幅において、5’−3’エキソヌクレアーゼ活性を有するポリメラーゼ酵素を使用する、請求項3又は4に記載の方法。
- 前記第一及び第二の蛍光部位は互いに、前記プローブ上に5個以下のヌクレオチドの範囲内で存在する、請求項4又は5に記載の方法。
- 前記SAプローブは、二次構造形成を可能とする核酸配列を含み、ここで、前記二次構造形成によって前記第一蛍光部位と第二蛍光部位との間が空間的に近接することとなる、請求項3〜6のいずれか1項に記載の方法。
- スタフィロコッカス・アウレウスの核酸を検出するためのキットであって:
− 配列番号2及び17〜20からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第一のオリゴヌクレオチド;
− 配列番号3、6、25及び26からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第二のオリゴヌクレオチド;並びに
− 配列番号4、27〜29及び32〜34からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、検出可能に標識された第三のオリゴヌクレオチド
を含むか、或いは、
− 配列番号8、12及び14〜16からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第一のオリゴヌクレオチド;
− 配列番号9及び21〜24からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、第二のオリゴヌクレオチド;並びに
− 配列番号10、30及び31からなる群から選択される配列又はその相補配列を含み、40個以下のヌクレオチドを有する、検出可能に標識された第三のオリゴヌクレオチド
を含む、前記キット。 - 前記検出可能に標識された第三のオリゴヌクレオチドが、ドナー蛍光部位及び対応するアクセプター蛍光部位を含む、請求項8に記載のキット。
- ヌクレオシド三リン酸、核酸ポリメラーゼ、及び前記核酸ポリメラーゼの機能のために必要なバッファーからなる群から選択される、少なくとも1つの追加のコンポーネントをさらに含む、請求項8又は9に記載のキット。
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CN103547685A (zh) | 2014-01-29 |
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CN103547685B (zh) | 2015-11-25 |
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US9816142B2 (en) | 2017-11-14 |
CA2831182C (en) | 2016-02-16 |
US20150218624A1 (en) | 2015-08-06 |
US20150218622A1 (en) | 2015-08-06 |
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US9816143B2 (en) | 2017-11-14 |
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