JP6783861B2 - 等温核酸増幅のためのレポーター染料、クエンチャーが含まれる等温ベースの二重機能性オリゴヌクレオチド及びそれを用いた核酸増幅並びに測定方法 - Google Patents
等温核酸増幅のためのレポーター染料、クエンチャーが含まれる等温ベースの二重機能性オリゴヌクレオチド及びそれを用いた核酸増幅並びに測定方法 Download PDFInfo
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Description
前記オリゴヌクレオチドのクエンチャーは、TAMRA、DABCYL、Black Hole Quencher 1もしくは2であるか、または500〜705nmの吸光波長帯であるものが好ましいが、これに限定されない。
前記オリゴヌクレオチドは、二本鎖から一本鎖に解離する融解温度が30〜70℃であることが好ましいが、これに限定されない。
前記方法の等温温度条件は50〜75℃の範囲であることが好ましいが、これに限定されない。
配列番号1:EB_B_F3
5’−GTGTGTTCAAGTACAGCATT−3’
配列番号2:EB_B_R3
5’−ATAAGGGAGGATGATCAAGG−3’
配列番号3:EB_B_FIP
5’−ACCTGGTGTTAGATGTTTATCTGAGGCCAAACATTATTTTGATAGCC−3’
配列番号4:EB_B_RIP
5’−TACATTAAGAGGAACCAATTTCCGCTGATAGAATTCCCACAATAAGTCTT−3’
配列番号5:EB_B_FIP_P2
5’−ACCTGG(internal dT−FAM)GTTAGATGTTTATCTGAGGCCAAACATTATTT(internal dT−BHQ1)GATAGCC−3’
配列番号6:EB_B_FIP_P2.5
5’−ACCTGGTGT(internal dT−FAM)AGATGTTTATCTGAGGCCAAACAT(internal dT−BHQ1)ATTTTGATAGCC−3’
配列番号7:EB_B_FIP_P3
5’−ACCTGGTGTTAGA(internal dT−FAM)GTTTATCTGAGGCCAAACAT(internal dT−BHQ1)ATTTTGATAGCC−3’
配列番号8:EB_B_RIP_P2
5’−TACA(internal dT−FAM)TAAGAGGAACCAATTTCCGCTGATAGAAT(internal dT−BHQ1)CCCACAATAAGTCTT−3’
配列番号9:EB_R_F3
5’−GCCTCACAATGTTAATCTTAGC−3’
配列番号10:EB_R_R3
5’−GATTGTCTCCCATGACCG−3’
配列番号11:EB_R_FIP
5’−CCTCTATGCCTCCTAAGTGCCAATCGAGAATATCCTCCTGAA−3’
配列番号12:EB_R_RIP
5’−GATTACAACAAAAACTGTGGACGAGCTGATCGTAACTTAAAACCAGT−3’
配列番号13:Res_LP
5’−GGTACGAACTCGGGC−3’
配列番号14:Res_RLP
5’−TGTGCACAAATCTCCTTAGT−3’
配列番号15:EB_R_FIP_P2
5’−CCTC(internal dT−FAM)ATGCCTCCTAAGTGCCAATCGAGAATATCC(internal dT−BHQ1)CCTGAA−3’
配列番号16:EB_R_RIP_P2
5’−GAT(internal dT−FAM)ACAACAAAAACTGTGGACGAGCTGATCGTAAC(internal dT−BHQ1)TAAAACCAGT−3’
配列番号17:EB_B_LP_F
5’−ATTACACTATACCATGACCCTT−3’
配列番号18:EB_B_LP_R
5’−CACTGCCTATGATTAAAGACT−3’
配列番号19:EB_B_FIP_P2−Q2_55
5’−CCTCAGATAAACATCTAAC−BHQ1−3’
配列番号20:EB_B_FIP_P2−Q2_60
5’−GGCCTCAGATAAACATCTAAC−BHQ1−3’
配列番号21:EB_B_FIP_P2−Q2_65
5’−TGTTTGGCCTCAGATAAACATCTAAC−BHQ1−3’
配列番号22:EB_B_FIP_P2−Q2_70
5’−GGCTATCAAAATAATGTTTGGCCTCAGATAAACATCTAAC−BHQ1−3’
配列番号23:EB_B_RIP_P2−Q_60
5’−CGGAAATTGGTTCCTCTTA−BHQ1−3’
Claims (17)
- ターゲット遺伝子の特定の配列に対するループ媒介等温核酸増幅反応(LAMP)用のフォワードインナープライマー(Forward Inner Primer)及びリバースインナープライマー(Reverse Inner Primer)のオリゴヌクレオチドであって、前記プライマーは2つのターゲット遺伝子部位を有し、前記2つのターゲット遺伝子部位のうち、5’末端へのターゲット遺伝子に対するプライマーの配列のうちの1つの配列にレポーター染料を付着させ、3’末端へのターゲット遺伝子に対するプライマー配列のうちの1つの配列にクエンチャーを付着させ、当該レポーター染料又はクエンチャーが存在している塩基は、アミノ-C6 (又はC2)-デオキシチミジン、アミノ-C6-デオキシアデノシン、アミノ-C6-デオキシシチジン、アミノ-C6-デオキシグアノシン、アミノ-C6-デオキシウリジン、またはアミノ-C6-デオキシリボースに置換され、配列の長さが21〜33mer以内であり、プライマーとプローブの特性を同時に有する、オリゴヌクレオチド。
- 前記オリゴヌクレオチドは、フォワードインターナルプローブとリバースインターナルプローブを個別又は共に使用することを特徴とする、請求項1に記載のオリゴヌクレオチド。
- 前記オリゴヌクレオチドのレポーター染料は、FAM、TET、HEX、TAMRA、ROX、TEXAS RED、CY3、及びCY5のうちの1つであるか、または450〜685nmの発光波長帯の染料であることを特徴とする、請求項1に記載のオリゴヌクレオチド。
- 前記オリゴヌクレオチドのクエンチャーは、TAMRA、DABCYL、Black Hole Quencher(登録商標) 1もしくは2であるか、または500〜705nmの吸光波長帯のクエンチャーであることを特徴とする、請求項1に記載のオリゴヌクレオチド。
- 前記オリゴヌクレオチドは、二本鎖から一本鎖に解離する融解温度が30〜70℃であることを特徴とする、請求項1に記載のオリゴヌクレオチド。
- 前記オリゴヌクレオチドは、配列番号5〜8、及び配列番号15〜16に記載されたオリゴヌクレオチドのうちの1つであることを特徴とする、請求項1に記載のオリゴヌクレオチド。
- 請求項1に記載のオリゴヌクレオチドを使用して等温でリアルタイム核酸増幅蛍光検出のためのループ媒介等温核酸増幅反応(LAMP)又は逆転写(RT)−LAMP反応を行う方法。
- 請求項1に記載のオリゴヌクレオチドを使用して等温又は2つ以上の温度条件で終点(end−point)核酸増幅蛍光検出のためのループ媒介等温核酸増幅反応(LAMP)又は逆転写(RT)−LAMP反応を行う方法。
- 前記オリゴヌクレオチドは、フォワードインターナルプローブとリバースインターナルプローブを個別又は共に使用することを特徴とする、請求項7又は8に記載の方法。
- 前記方法の等温温度条件は50〜75℃の範囲であることを特徴とする、請求項7又は8に記載の方法。
- 前記方法は、DNA及びcDNA核酸を対象として行うことを特徴とする、請求項7又は8に記載の方法。
- 前記方法は、RNA核酸を対象として逆転写反応後に特定の遺伝子をワンステップ反応で行うことを特徴とする、請求項7又は8に記載の方法。
- 請求項1に記載のオリゴヌクレオチドを含む感染性疾患、遺伝性疾患、薬剤耐性、薬物抵抗性または感受性検体に対する特定の遺伝子を個別又は同時多重的に増幅するキット。
- 請求項1に記載のオリゴヌクレオチドを有効成分として含む等温核酸増幅反応用組成物。
- 前記等温核酸増幅反応用組成物は、配列番号5〜8、及び配列番号15〜16に記載されたオリゴヌクレオチドのうちの1つを含むことを特徴とする、請求項14に記載の等温核酸増幅反応用組成物。
- 請求項1に記載のオリゴヌクレオチドを有効成分として含むエボラウイルス(Ebola virus)核酸増幅用組成物。
- 請求項1に記載のオリゴヌクレオチドとアンチセンスプローブを共に使用して、DNA、RNA、またはcDNAに対する等温核酸増幅反応を行う方法。
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