JPWO2020072409A5 - - Google Patents
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- JPWO2020072409A5 JPWO2020072409A5 JP2021542076A JP2021542076A JPWO2020072409A5 JP WO2020072409 A5 JPWO2020072409 A5 JP WO2020072409A5 JP 2021542076 A JP2021542076 A JP 2021542076A JP 2021542076 A JP2021542076 A JP 2021542076A JP WO2020072409 A5 JPWO2020072409 A5 JP WO2020072409A5
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- 230000003321 amplification Effects 0.000 claims description 78
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 78
- 239000000523 sample Substances 0.000 claims description 72
- 238000001514 detection method Methods 0.000 claims description 59
- 239000000203 mixture Substances 0.000 claims description 42
- 150000007523 nucleic acids Chemical group 0.000 claims description 27
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 24
- 241000701085 Human alphaherpesvirus 3 Species 0.000 claims description 22
- 239000007850 fluorescent dye Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000009396 hybridization Methods 0.000 claims description 8
- 238000010791 quenching Methods 0.000 claims description 8
- 230000000171 quenching effect Effects 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims description 4
- 102100034343 Integrase Human genes 0.000 claims description 4
- 108091034117 Oligonucleotide Proteins 0.000 claims description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- -1 dNTPs Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
Description
「安定な」または「検出に対して安定な」という用語は、核酸二本鎖が変性する温度よりも低い反応混合物の温度を示す。
特定の実施形態では、例えば以下の項目が提供される。
（項目１）
水痘帯状疱疹ウイルス（ＶＺＶ）標的核酸配列を増幅するための組成物であって、（ａ）配列番号３８もしくはその相補体または配列番号３９もしくはその相補体に存在する１９～２３個のヌクレオチドの配列に対して少なくとも９０％の同一性を有する１９～２３個の連続核酸塩基を含む１９～５０核酸塩基の長さの順方向増幅プライマー、および（ｂ）配列番号３８もしくはその相補体または配列番号３９もしくはその相補体に存在する１９～２３個のヌクレオチドの配列に対して少なくとも９０％の同一性を有する１９～２３個の連続核酸塩基を含む１９～５０核酸塩基の長さの逆方向増幅プライマーを含む組成物。
（項目２）
前記順方向増幅プライマーが、配列番号１、２、３、４、５、６、７、２３、２４、２５、２６、または２７の核酸塩基配列を含む、項目１に記載の組成物。
（項目３）
前記逆方向増幅プライマーが、配列番号１６、１７、１８、１９、２０、２１、２２、３４、３５、３６または３７の核酸塩基配列を含む、項目１～２のいずれか一項に記載の組成物。
（項目４）
（ａ）前記順方向増幅プライマーが、配列番号１の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１６の核酸塩基配列を含み、
（ｂ）前記順方向増幅プライマーが、配列番号１の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１７の核酸塩基配列を含み、
（ｃ）前記順方向増幅プライマーが、配列番号２の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１７の核酸塩基配列を含み、
（ｄ）前記順方向増幅プライマーが、配列番号３の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１８の核酸塩基配列を含み、
（ｅ）前記順方向増幅プライマーが、配列番号４の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１９の核酸塩基配列を含み、
（ｆ）前記順方向増幅プライマーが、配列番号５の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号２０の核酸塩基配列を含み、
（ｇ）前記順方向増幅プライマーが、配列番号６の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号２１の核酸塩基配列を含み、
（ｈ）前記順方向増幅プライマーが、配列番号７の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号２２の核酸塩基配列を含み、
（ｉ）前記順方向増幅プライマーが、配列番号２３の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３４の核酸塩基配列を含み、
（ｊ）前記順方向増幅プライマーが、配列番号２４の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３４の核酸塩基配列を含み、
（ｋ）前記順方向増幅プライマーが、配列番号２５の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３５の核酸塩基配列を含み、
（ｌ）前記順方向増幅プライマーが、配列番号２６の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３６の核酸塩基配列を含み、または
（ｍ）前記順方向増幅プライマーが、配列番号２７の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３７の核酸塩基配列を含む、項目１～３のいずれか一項に記載の組成物。
（項目５）
増幅された水痘帯状疱疹ウイルス（ＶＺＶ）標的核酸配列を検出するための検出プローブをさらに含み、前記検出プローブが、少なくとも１つの検出可能な標識を含む、項目１～４のいずれか一項に記載の組成物。
（項目６）
前記検出プローブが、配列番号８、９、１０、１１、１２、１３、１４、１５、２８、２９、３０、３１、３２、または３３の核酸塩基配列を含む、項目５に記載の組成物。
（項目７）
（ａ）前記検出プローブが、配列番号８もしくは９の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号１の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１６もしくは１７の核酸塩基配列を含み、
（ｂ）前記検出プローブが、配列番号９の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号２の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１７の核酸塩基配列を含み、
（ｃ）前記検出プローブが、配列番号１０の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号３の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１８の核酸塩基配列を含み、
（ｄ）前記検出プローブが、配列番号１１もしくは１２の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号４の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号１９の核酸塩基配列を含み、
（ｆ）前記検出プローブが、配列番号１３の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号５の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号２０の核酸塩基配列を含み、
（ｇ）前記検出プローブが、配列番号１４の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号６の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号２１の核酸塩基配列を含み、
（ｈ）前記検出プローブが、配列番号１５の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号７の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号２２の核酸塩基配列を含み、
（ｉ）前記検出プローブが、配列番号２８の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号２３もしくは２４の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３４の核酸塩基配列を含み、
（ｊ）前記検出プローブが、配列番号２９もしくは３０の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号２５の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３５の核酸塩基配列を含み、
（ｋ）前記検出プローブが、配列番号３１の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号２６の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３６の核酸塩基配列を含み、または
（ｌ）前記検出プローブが、配列番号３２もしくは３３の核酸塩基配列を含み、前記順方向増幅プライマーが、配列番号２７の核酸塩基配列を含み、前記逆方向増幅プライマーが、配列番号３７の核酸塩基配列を含む、項目６に記載の組成物。
（項目８）
前記順方向増幅プライマー、前記逆方向増幅プライマー、および／または前記検出プローブが、少なくとも１つの修飾ヌクレオチドを含む、項目１～７のいずれか一項に記載の組成物。
（項目９）
前記修飾ヌクレオチドが、２’－Ｏ－メチル修飾ヌクレオチド、２’－フルオロ修飾ヌクレオチド、または５－メチルシトシンを含む、項目８に記載の組成物。
（項目１０）
前記少なくとも１つの検出可能な標識が、
（ａ）化学発光標識、
（ｂ）蛍光標識、
（ｃ）消光剤、または
（ｄ）（ａ）、（ｂ）、および（ｃ）のうちの２つ以上の組み合わせ、からなる群から選択される、項目５～９のいずれか一項に記載の組成物。
（項目１１）
前記少なくとも１つの検出可能な標識が、前記蛍光標識、前記消光剤、または前記蛍光標識および前記消光剤の両方を含む、項目１０に記載の組成物。
（項目１２）
前記検出プローブが、前記検出プローブの３’末端と塩基対合する５’非標的ハイブリッド形成配列、または前記検出プローブの５’末端と塩基対合する３’非標的ハイブリッド形成配列を含む、項目１～１１のいずれか一項に記載の組成物。
（項目１３）
前記検出プローブが、分子ビーコンまたは分子トーチを含む、項目１２に記載の組成物。
（項目１４）
緩衝液、塩、ｄＮＴＰ、界面活性剤、および酵素のうちの１種以上をさらに含む、項目１～１３のいずれか一項に記載の組成物。
（項目１５）
前記酵素が、熱安定性ＤＮＡポリメラーゼ、逆転写酵素、ＲＮＡポリメラーゼ、または熱安定性ＤＮＡポリメラーゼ、逆転写酵素、およびＲＮＡポリメラーゼのうちの任意の２種以上の組み合わせを含む、項目１４に記載の組成物。
（項目１６）
前記増幅プライマーが、水溶液中に存在するか、凍結されているか、または凍結乾燥されている、項目１～１５のいずれか一項に記載の組成物。
（項目１７）
前記組成物が、２対以上の増幅プライマーおよび／または２つ以上の検出プローブを含み、増幅プライマーの各対が、順方向増幅プライマーおよび逆方向増幅プライマーからなる、項目１～１６のいずれか一項に記載の組成物。
（項目１８）
前記２対以上の増幅プライマーおよび／または２つ以上の検出プローブが、同じまたは異なる生物の標的核酸配列を増幅する、項目１７に記載の組成物。
（項目１９）
内部標準の標的核酸配列、内部標準の標的核酸配列を増幅および／もしくは検出するためのオリゴマー、またはこれらの組み合わせをさらに含む、項目１～１８のいずれか一項に記載の組成物。
（項目２０）
配列番号８、９、１０、１１、１２、１３、１４、１５、２８、２９、３０、３１、３２、または３３の核酸塩基配列を含むオリゴヌクレオチドを含む、ＶＺＶ標的核酸配列を検出するための検出プローブであって、前記オリゴヌクレオチドが、１つ以上の検出可能な標識を含む、検出プローブ。
（項目２１）
前記検出プローブが、少なくとも１つの修飾ヌクレオチドを含む、項目２０に記載の検出プローブ。
（項目２２）
前記修飾ヌクレオチドが、２’－Ｏ－メチル修飾ヌクレオチド、２’－フルオロ修飾ヌクレオチド、または５－メチルシトシンを含む、項目２１に記載の検出プローブ。
（項目２３）
前記検出可能な標識のうちの１つ以上が、
（ａ）化学発光標識、
（ｂ）蛍光標識、
（ｃ）消光剤、または
（ｄ）（ａ）、（ｂ）、および（ｃ）のうちの２つ以上の組み合わせ、からなる群から選択される、項目２０～２２のいずれか一項に記載の検出プローブ。
（項目２４）
前記検出可能な標識のうちの１つ以上が、前記蛍光標識、前記消光剤、または前記蛍光標識および前記消光剤の両方を含む、項目２３に記載の検出プローブ。
（項目２５）
前記検出プローブが、前記検出プローブの３’末端と塩基対合する５’非標的ハイブリッド形成配列、または前記検出プローブの５’末端と塩基対合する３’非標的ハイブリッド形成配列を含む、項目２０～２４のいずれか一項に記載の検出プローブ。
（項目２６）
前記検出プローブが、分子ビーコンまたは分子トーチを含む、項目２５に記載の検出プローブ。
（項目２７）
ＶＺＶ標的核酸配列を増幅するための方法であって、
（ａ）ＶＺＶ標的核酸配列を含むまたは含む疑いがある試料を入手することと、
（ｂ）前記試料を項目１～１９のいずれか一項に記載の組成物と接触させることと、
（ｃ）前記標的核酸配列を増幅するのに十分な条件を提供することにより、前記ＶＺＶ標的核酸配列が前記試料中に存在する場合、前記ＶＺＶ標的核酸配列の増幅産物を生成することと、を含む方法。
（項目２８）
前記方法が、前記増幅産物の存否を決定するために、前記試料と項目２０～２６のいずれか一項に記載の検出プローブとを接触させることをさらに含む、項目２７に記載の方法。
（項目２９）
試料中のＶＺＶの存否を決定するための方法であって、
（ａ）ＶＺＶ標的核酸配列を含むまたは含む疑いがある試料を入手することと、
（ｂ）前記試料と項目１～１９のいずれか一項に記載の組成物とを接触させることと、
（ｃ）前記標的核酸配列を増幅するのに十分な条件を提供することによって増幅産物を生成することと、
（ｄ）前記増幅産物の存否を検出することと、を含む方法。
The terms "stable" or "stable to detection" refer to temperatures of the reaction mixture below the temperature at which nucleic acid duplexes denature.
In certain embodiments, for example, the following items are provided.
(Item 1)
A composition for amplifying a varicella zoster virus (VZV) target nucleic acid sequence comprising: (a) a sequence of 19-23 nucleotides present in SEQ ID NO: 38 or its complement or SEQ ID NO: 39 or its complement and (b) a forward amplification primer 19-50 nucleobases in length comprising 19-23 contiguous nucleobases having at least 90% identity to SEQ ID NO:38 or its complement or or a reverse amplification primer 19-50 nucleobases in length comprising 19-23 contiguous nucleobases having at least 90% identity to a sequence of 19-23 nucleotides present in its complement; A composition comprising:
(Item 2)
The composition of item 1, wherein the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 23, 24, 25, 26, or 27.
(Item 3)
3. The composition of any one of items 1-2, wherein the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 34, 35, 36 or 37. thing.
(Item 4)
(a) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 1 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 16;
(b) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 1 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 17;
(c) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:2 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:17;
(d) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:3 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:18;
(e) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 4 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 19;
(f) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:5 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:20;
(g) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:6 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:21;
(h) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:7 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:22;
(i) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:23 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:34;
(j) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:24 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:34;
(k) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:25 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:35;
(l) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:26 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:36; or
(m) The composition of any one of items 1 to 3, wherein the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:27 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:37. thing.
(Item 5)
5. Any one of items 1-4, further comprising a detection probe for detecting the amplified varicella zoster virus (VZV) target nucleic acid sequence, said detection probe comprising at least one detectable label. composition.
(Item 6)
6. The composition of item 5, wherein the detection probe comprises the nucleobase sequence of SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 28, 29, 30, 31, 32, or 33.
(Item 7)
(a) the detection probe comprises the nucleobase sequence of SEQ ID NO: 8 or 9, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 1, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 16 or 17; comprising a nucleobase sequence;
(b) the detection probe comprises the nucleobase sequence of SEQ ID NO: 9, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 2, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 17; including
(c) the detection probe comprises the nucleobase sequence of SEQ ID NO: 10, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 3, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 18; including
(d) the detection probe comprises the nucleobase sequence of SEQ ID NO: 11 or 12, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 4, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 19; contains an array,
(f) the detection probe comprises the nucleobase sequence of SEQ ID NO: 13, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 5, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 20; including
(g) the detection probe comprises the nucleobase sequence of SEQ ID NO: 14, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 6, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 21; including
(h) the detection probe comprises the nucleobase sequence of SEQ ID NO: 15, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 7, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 22; including
(i) the detection probe comprises the nucleobase sequence of SEQ ID NO: 28, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 23 or 24, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 34; contains an array,
(j) the detection probe comprises the nucleobase sequence of SEQ ID NO: 29 or 30, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 25, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 35; contains an array,
(k) the detection probe comprises the nucleobase sequence of SEQ ID NO: 31, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 26, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 36; includes, or
(l) the detection probe comprises the nucleobase sequence of SEQ ID NO: 32 or 33, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO: 27, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO: 37; A composition according to item 6, comprising a sequence.
(Item 8)
Composition according to any one of items 1 to 7, wherein said forward amplification primer, said reverse amplification primer and/or said detection probe comprise at least one modified nucleotide.
(Item 9)
9. The composition of item 8, wherein said modified nucleotides comprise 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or 5-methylcytosine.
(Item 10)
wherein the at least one detectable label is
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher, or
(d) A composition according to any one of items 5 to 9, selected from the group consisting of (a), (b), and a combination of two or more of (c).
(Item 11)
11. The composition of item 10, wherein said at least one detectable label comprises said fluorescent label, said quenching agent, or both said fluorescent label and said quenching agent.
(Item 12)
Item 1, wherein the detection probe comprises a 5' non-target hybridization sequence that base-pairs to the 3' end of the detection probe, or a 3' non-target hybridization sequence that base-pairs to the 5' end of the detection probe. 12. The composition of any one of claims 1-11.
(Item 13)
13. The composition of item 12, wherein said detection probe comprises a molecular beacon or a molecular torch.
(Item 14)
14. The composition of any one of items 1-13, further comprising one or more of buffers, salts, dNTPs, surfactants and enzymes.
(Item 15)
15. The composition of item 14, wherein the enzyme comprises a thermostable DNA polymerase, reverse transcriptase, RNA polymerase, or a combination of any two or more of thermostable DNA polymerase, reverse transcriptase, and RNA polymerase. thing.
(Item 16)
16. The composition of any one of items 1-15, wherein said amplification primers are in aqueous solution, frozen or lyophilized.
(Item 17)
17. Any one of items 1-16, wherein the composition comprises two or more pairs of amplification primers and/or two or more detection probes, each pair of amplification primers consisting of a forward amplification primer and a reverse amplification primer. 13. The composition of claim 1.
(Item 18)
18. The composition of item 17, wherein said two or more pairs of amplification primers and/or two or more detection probes amplify target nucleic acid sequences of the same or different organisms.
(Item 19)
Composition according to any one of items 1 to 18, further comprising an internal control target nucleic acid sequence, an oligomer for amplifying and/or detecting the internal control target nucleic acid sequence, or a combination thereof.
(Item 20)
for detecting a VZV target nucleic acid sequence comprising an oligonucleotide comprising the nucleobase sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 28, 29, 30, 31, 32, or 33 A detection probe, wherein said oligonucleotide comprises one or more detectable labels.
(Item 21)
21. The detection probe of item 20, wherein said detection probe comprises at least one modified nucleotide.
(Item 22)
22. The detection probe of item 21, wherein said modified nucleotides comprise 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or 5-methylcytosine.
(Item 23)
one or more of the detectable labels are
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher, or
(d) the detection probe of any one of items 20-22, selected from the group consisting of (a), (b), and a combination of two or more of (c).
(Item 24)
24. The detection probe of item 23, wherein one or more of said detectable labels comprises said fluorescent label, said quenching agent, or both said fluorescent label and said quenching agent.
(Item 25)
Item 20, wherein said detection probe comprises a 5' non-target hybridization sequence that base-pairs to the 3' end of said detection probe, or a 3' non-target hybridization sequence that base-pairs to the 5' end of said detection probe. 25. A detection probe according to any one of claims 1-24.
(Item 26)
26. The detection probe of item 25, wherein said detection probe comprises a molecular beacon or a molecular torch.
(Item 27)
A method for amplifying a VZV target nucleic acid sequence comprising:
(a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence;
(b) contacting the sample with the composition of any one of items 1-19;
(c) providing conditions sufficient to amplify said target nucleic acid sequence, thereby producing an amplification product of said VZV target nucleic acid sequence when said VZV target nucleic acid sequence is present in said sample; How to include.
(Item 28)
28. The method of item 27, wherein the method further comprises contacting the sample with a detection probe of any one of items 20-26 to determine the presence or absence of the amplification product.
(Item 29)
A method for determining the presence or absence of VZV in a sample, comprising:
(a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence;
(b) contacting the sample with the composition of any one of items 1-19;
(c) producing an amplification product by providing conditions sufficient to amplify said target nucleic acid sequence;
(d) detecting the presence or absence of said amplification product.
Claims (25)
（ａ）配列番号４の核酸塩基配列を含む順方向増幅プライマー、および
（ｂ）配列番号１９の核酸塩基配列を含む逆方向増幅プライマー
を含む組成物。 A composition for amplifying a varicella zoster virus (VZV) target nucleic acid sequence comprising:
(a) a forward amplification primer comprising the nucleobase sequence of SEQ ID NO: 4 , and
(b) a composition comprising a reverse amplification primer comprising the nucleobase sequence of SEQ ID NO:19 ;
（ａ）化学発光標識、
（ｂ）蛍光標識、
（ｃ）消光剤、または
（ｄ）（ａ）、（ｂ）、および（ｃ）のうちの２つ以上の組み合わせ、からなる群から選択される、請求項２～５のいずれか一項に記載の組成物。 wherein the at least one detectable label is
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher; or (d) a combination of two or more of ( a), (b), and (c). The described composition.
（ａ）化学発光標識、
（ｂ）蛍光標識、
（ｃ）消光剤、または
（ｄ）（ａ）、（ｂ）、および（ｃ）のうちの２つ以上の組み合わせ、からなる群から選択される、請求項１６～１８のいずれか一項に記載の検出プローブ。 one or more of the detectable labels are
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher; or ( d ) a combination of two or more of (a), (b), and (c). Detection probes as described.
（ａ）ＶＺＶ標的核酸配列を含むまたは含む疑いがある試料を入手することと、
（ｂ）前記試料を請求項１～１５のいずれか一項に記載の組成物と接触させることと、
（ｃ）前記標的核酸配列を増幅するのに十分な条件を提供することにより、前記ＶＺＶ標的核酸配列が前記試料中に存在する場合、前記ＶＺＶ標的核酸配列の増幅産物を生成することと、を含む方法。 A method for amplifying a VZV target nucleic acid sequence comprising:
(a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence;
(b) contacting the sample with the composition of any one of claims 1-15 ;
(c) providing conditions sufficient to amplify said target nucleic acid sequence, thereby producing an amplification product of said VZV target nucleic acid sequence when said VZV target nucleic acid sequence is present in said sample; How to include.
（ａ）ＶＺＶ標的核酸配列を含むまたは含む疑いがある試料を入手することと、
（ｂ）前記試料と請求項１～１５のいずれか一項に記載の組成物とを接触させることと、
（ｃ）前記標的核酸配列を増幅するのに十分な条件を提供することによって増幅産物を生成することと、
（ｄ）前記増幅産物の存否を検出することと、を含む方法。
A method for determining the presence or absence of VZV in a sample, comprising:
(a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence;
(b) contacting the sample with the composition of any one of claims 1-15 ;
(c) producing an amplification product by providing conditions sufficient to amplify said target nucleic acid sequence;
(d) detecting the presence or absence of said amplification product.
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