1286159 五、發明說明（1) 本發明係關於檢棒（d i p s t i c k s )之增強核酸偵測。本發 明之檢棒用於偵測樣品溶液中目標核酸之存在，例如鑑定 病人是否遭受引起疾病之微生物，例如砂眼披衣菌 {Chlamydia trachomatis、的感染。 用於偵測樣品溶液中目標核酸存在之一些慣用測試，是 仰賴利用聚合酶連鎖反應（PCR)之目標核酸的增幅。此反 應可偵測小量的目標核酸，然而得到結果前需耗時數小時 ，但因通常希望可盡快得到結果，例如維持病人的等待時 間減至最短，故此爲其顯著缺點。此等方法之進一步缺點 是需要昂貴的專家技術進行反應以及柑當高成本的試劑。 相較之下，檢棒可偵測未放大的目標核酸，不需任何專 家技術且能比PCR爲基礎之方法更快速得到結果。病人可 於同一訪視中處理，故特別有益於不喜歡或不能曰後複診 的病人。 敘述於US 5,3 1 0,650之典型慣用檢棒中，單股DNA捕 獲探針（c a p t u r e p r 〇 b e )是固定於遠離濾、紙一末端（接觸端） 之捕獲區的硝基纖維濾紙上。捕獲探針的部分序列互補於 欲測目標核酸第一區域之序列。標識之單股DNA偵測探針 (d e t e c t i ο n p r 〇 b e )是固定在位於濾紙捕獲區及接觸端間 之探針區的硝基纖維濾紙上。偵測探針含有互補於目標核 酸第二區域序列（不同於第一區域）之序列。 在認爲含有目標DNA之樣品溶液中偵測目標DNA時’將 硝基纖維濾紙的接觸端接觸樣品溶液。樣品溶液經由毛細 1286159 五、發明說明（2) 作用在濾紙上移並經過探針區及捕獲區。當樣品溶液經過 探針區時，可使偵測探針移動並導致其與樣品溶液一起朝 向捕獲區上升。然後移動的偵測探針能雜交到樣品溶液中 存在之任何目標DNA的第二區域。 當雜交之偵測探針及目標DNA到達捕獲區時，目標DNA 的第一區域能雜交到固定的捕獲探針。藉此在目標核酸、 捕獲探針及標識之偵測探針之間形成三級複合物。捕獲區 出現標識時代表樣品溶液中存在目標DNA。 至於第二類型之慣用檢棒，標識之DNA偵測探針未固定 在硝基纖維濾紙上。而是在允許偵測探針雜交到樣品溶液 中任何目標核酸之條件下加入偵測探針。然後將硝基纖維 濾紙的接觸端接觸樣品溶液，且當樣品溶液在檢棒上移時 ’雜交到偵測探針之目標核酸可經由捕獲探針在捕獲區被 捕獲。 然而發現慣用檢棒之核酸偵測的敏感度低。若目標核酸 爲雙股時，核酸偵測的敏感度特低。因此，有時可能樣品 溶液中存在之目標核酸未被偵測到。因此希望改良檢棒之 核酸偵測的敏感度。 根據本發明第一部分提供一種檢棒用於測試樣品溶液中 目標核酸之存在，檢棒含有： 一種層析片具有接觸端用於接觸樣品溶液；以及 一種捕獲探針，固定在遠離接觸端之捕獲區上，捕獲探 針能雜交到目標核酸或已結合目標核酸之吊鉤捕獲探針 -4- 1286159 五、發明說明 ( 3) (hook c ap t u r e probe)，其中 捕獲探針的一或兩末端 連 接 一或 更 多 核 苷 酸，較佳至少三 個核苷酸，其當捕獲探 針 雜 交到 巨 標 核 酸 時不會雜交到目 標核酸，或當捕獲探針 雜 交 到吊 鉤 捕 獲 探 針時不會雜交到 吊鉤捕獲探針。 根 據 本 發 明 第一部分也提供 一種套組，用於測試樣 品 溶 液中 巨 標 核 酸 存在之，其包含 根據本發明之檢棒，以 及 一 種偵 測 探 針 能 雜交到目標核酸 ，藉此運用偵測探針而 偵 測 目標 核 酸 0 文 中 使 用 之 “層析片”一詞 意指能藉由毛細作用傳 送 溶 液之任何可 浸 透片材料。 偵 測 探 針 可改爲可釋放性地 固定在位於檢棒接觸端 與 捕 獲區 間 之 探 針 區° 偵 測 探 針 的 一或兩末端可連 接一或更多核苷酸，較 佳 至 少三 個 核 苷 酸 ，其當偵測探針 雜交到目標核酸時不會 雜 交 到目 標 核 酸 〇 根 據 本 發 明 第二部分，也提 供一種檢棒用於測試樣 品 溶 液中 巨 標 核 酸 之存在，其包括 : 一 種 層 析片 具有接觸端，用: 於接觸樣品溶液； 一 種 捕 獲 部 分（capture mo i ety)固定在遠離接觸端 之 捕 獲區 捕 獲 部 分能直接或間接; 結合目標核酸；以及 一 種 偵 測 探 針，可釋放性地 固定在接觸端與捕獲區 間 之 探針 區 y 偵 測 探針能雜交到目 標核酸，藉此運用偵測 探 針 而偵測目 標 核 酸，其中偵測探 -5- 針的一或兩末端可連接 或 1286159 五、 發明說明 (4) 更 多 核 苷 酸 ，較佳至 少 三 個 核 苷酸， 其 當 偵測探針雜 交 到 巨 標 核 酸 時 不會雜交 到 巨 標 核 酸。 根 據 本 發 明更進一 步 提 供 — 種套組 用 於 測試樣品溶 液 中 巨 標 核 酸 之存在，其包含 ·· 根 據 本 發 明第二部 分 之 檢 棒 ，其中 捕 獲 部分能結合 到 捕 獲 配位 體 (其已連接到已結合目標核酸之捕獲探針）；， 以‘ 及 —* 種 捕 獲 探針能雜 交 到 巨 標 核酸或 已 結 合目標核酸 之 吊 鉤 捕 獲 探 針 ，其中捕 獲 探 針 連 接到捕 獲 配 位體其能經 由 捕 獲 部 分 結 合 〇 捕 獲 探 針 的一或兩 末 端 可 連 接一或 更 多 核苷酸，較 佳 至 少 二 個 核 苷 酸，其當 捕 獲 探 針 雜交到 巨 標 核酸時不會 雜 交 到 巨 標 核 酸 ，或當捕 獲 探 針 雜 交到吊 鉤 捕 獲探針時不 會 雜 交 到吊 鉤 捕 獲探針。 根 據 本 發 明也提供 — 種 套 組 用於測 試 樣 品溶液中目 標 核 酸 之存在 其包含： i ) 一 種 檢 棒，含有 層 析片 具 有接觸 端 用 於接觸樣品 溶 液 以 及 — 種 捕獲探針 能 雜 交 到 目標核 酸 或 已結合目標 核 酸 之 吊 鉤 捕 獲 探針，而 捕 獲 探 針 固定在 層 析 片遠離接觸 觸 之 捕 獲 1¾ 上 以及 ϋ)- -種偵測探針能雜交到1 目標核ί 駿 ，藉此運用偵 測 探 針而 偵 測目 標核酸， 其 中 偵 測 探針的 一 或 兩末端可連 接 —* 或 更 多 核 苷 酸，較佳 至 少 三 個 核苷酸 ， 其 當偵測探針 雜 交 到目 標 核 酸 時不會雜 父 到 巨' 標; • 6 - 核酸。 1286159 五、發明說明（5 ) 根據本發明也提供一種套組用於測試樣品溶液中目標核 酸之存在，其包含： i ) 一種檢棒，含有層析片具有接觸端用於接觸樣品溶液 ，以及一種捕獲部分固定在層析片遠離接觸端之捕獲區， 捕獲部分能直接或間接結合目標核酸； i i ) 一種捕獲探針能雜交到目標核酸或已結合目標核酸 之吊鉤捕獲探針，其中捕獲探針連接捕獲配位體而能經由 捕獲部分結合；以及 i i i ) 一種偵測探針能雜交到目標核酸，其中偵測探針的 一或兩末端可連接一或更多核苷酸，較佳至少三個核苷酸 ，其當偵測探針雜交到目標核酸時不會雜交到目標核酸。 根據本發明也提供一種套組用於測試樣品溶液中目標核 酸之存在，其包含： i)一種檢棒，含有層析片具有接觸端用於接觸樣品溶液 ，以及一種捕獲部分固定在層析片遠離接觸端之捕獲區； 1 1 ) 一種捕獲探針能雜交到目標核酸或已結合目標核酸 之吊鉤捕獲探針，其中捕獲探針連接捕獲配位體而能經由 捕獲部分結合，以及捕獲探針的一或兩末端可連接一或更 多核苷酸，較佳至少三個核苷酸，其當捕獲探針雜交到目 標核酸時不會雜交到目標核酸；以及 i i i ) 一種偵測探針能雜交到目標核酸，藉此運用偵測探 針而偵測目標核酸。 本發明檢棒及套組可用於測試樣品溶液中存在目標核酸 1286159 五、 發明說明 ( 6 ) 之 方 法 中 其 中 能 雜 交到目標核酸之偵測探針在偵測 探 針 能 雜 交 到 巨 標 核 酸 之 條件下與樣品溶液培育。檢棒的 接 觸 Λ山 觸 接 觸 樣 品 溶 液 使 得樣品溶液經由毛細作用於檢棒 向 上 移 行 0 然 後 在 樣 品 溶 液雜交到偵測探針之目標核酸能 在 捕 獲 T& 經 由 捕 獲 探 針 捕 獲。樣品溶液中目標核酸之存在 可 由 捕 獲 區 出現 偵 測 探 針指示出來。 然 而 偵 測 探 針 可 釋 放性地固定在檢棒，例如位在層 析 片 接 觸 端 與 捕 獲 區 間 之 探針區，將檢棒接觸端與樣品溶 液 接 觸 以 至 樣 品 溶 液 經 由 毛細作用於檢棒向上移行。當樣 品 溶 液 通 ^JEL 過 檢 棒 探 針 區 時使偵測探針流動，以至偵測探針 能 雜 交 到 樣 品 溶 液 中 之 巨 標核酸並與目標核酸移行到捕獲 區 〇 當 樣 品 溶 液 通 n'JH, m 捕 獲 區時，雜交到偵測探針之目標核 酸 經 由 捕 獲 探 針 被 捕 獲 Ο 爾後樣品溶液中目標核酸之存在 可 由 捕 獲 區 出現 偵 測 探 針指示出來。 捕 獲 部 分 可 經 由 鹼 基配對或非鹼基配對之交互作用 直 接 或 間 接 結 合 巨 標 核 酸。舉例而言，捕獲部分可含有 捕 獲 探 針 其 能 直 接 雜 交 到目標核酸。另外，捕獲部分可含有 捕 獲 探 針 > 其 能 直 接 雜交到已結合目標核酸之吊鉤捕 獲 探 針 〇 捕 獲 部 分 可 能 結 合 到捕獲配位體（已連接到已結合 g 標 核 酸 之 捕 獲 探 針 ) ，而藉此間接將捕獲部分結合到目 標 核 酸 〇 舉 例 而 言 j 捕 獲 部分可能是抗體或抗體片段。若 捕 獲 部 分 連 接 到 捕 獲 配 位 體，捕獲探針可能連接到一連接 -8- 捕 獲 1286159 五、發明說明（7) 配位體之捕獲探針間隔子（capture probe spacer)，而將 捕獲配位體與捕獲探針間隔。 吊鉤捕獲探針可加到樣品溶液，以至能結合到樣品溶液 中之目標核酸，並當樣品溶液經由毛細作用於檢棒向上移 行時由捕獲探針捕獲。 捕獲探針，吊鉤捕獲探針及偵測探針可各含有至少一個 核酸或核酸類似物。此處探針含有一以上核酸或核酸類似 物時，較佳是雜交在一起。 偵測探針可連接標識，而使能藉此在捕獲區直接偵測目 標核酸。另外偵測探針可連接偵測配位體而能利用偵測配 位體結合部分間接偵測目標核酸。適合的標識例如包括紡 織染料，金屬溶膠如金膠體，以及著色顆粒如著色乳膠顆 粒。此等標識能直接連接偵測探針，或者，若偵測探針連 接一偵測配位體時則直接連接到偵測配位體結合部分。 適合之捕獲或偵測配位體，舉例包括生物素（例如經由 抗生物素抗體，抗生物素蛋白（avidin)、鏈抗生物素蛋白 (s t r ep t a v i d i η )、或其衍生物捕獲或偵測），螢光素（例如 經由抗-螢光素抗體捕獲或偵測）以及2,4 -二硝基苯酚 (DNP )(例如經由抗-DNP抗體捕獲或偵測）。 偵測探針可含有一般偵測探針，其能雜交到已結合目標 核酸之吊鉤偵測探針。一般偵測探針可連接到標識或偵測 配位體而藉此偵測到偵測探針。 需知本發明套組可進一步含有使用套組偵測樣品溶液之 1286159 五、發明說明（8) 目標核酸所需之任何試劑。例如本發明套組可含有連接偵 測配位體之偵測探針，可進一步含有偵測配位體結合部分 〇 偵測配位體結合部分可含有抗體或抗體片段，或非抗體 。例如，若偵測配位體含有生物素，則偵測配位體結合部 分可含有抗生物素抗體，鏈抗生物素蛋白，抗生物素蛋白 或其保有生物素結合活性之衍生物。較佳爲偵測配位體結 合部分已標識，而使能藉此運用偵測探針及偵測配位體結 合部分間接偵測目標核酸。 需知本發明是關於連接一或更多“非配對”核苷酸之偵 測探針及/或捕獲探針之使用，以及此偵測探針或捕獲探 針可固定於檢棒或視目標核酸之偵測方法與樣品溶液培育 〇 根據本發明使用含有核鹼基之非配對核苷酸被認爲特別 有功效，係因核苷酸或核鹼基能與形成於捕獲探針或偵測 探針與目標核酸間之鹼基形成堆積交互作用。堆積交互作 用之形成被認爲可增強捕獲探針或偵測探針雜交到目標核 酸，藉此改良目標核酸在檢棒捕獲區的捕獲或偵測效率。 適合情況下，本發明檢棒及套組可用於下列形態之檢棒 分析測試樣品溶液中目標核酸之存在： 1 )提供一種檢棒，含有層析片具有接觸端及捕獲探針固 定在遠離接觸端之捕獲區上，捕獲探針能雜交到目標核酸 。一種偵測探針，在偵測探針能雜交到目標核酸之條件下 -10- 1286159 五、發明說明（9 ) 接觸樣品溶液。以檢棒的接觸端接觸樣品溶液，使得樣品 溶液經由毛細作用移行至捕獲區，藉此使目標核酸及偵測 探針與樣品溶液移行至捕獲區，並將目標核酸捕獲在捕獲 區。樣品溶液中目標核酸之存在可由捕獲區出現偵測探針 指示出來。 此分析之變化中，偵測探針可釋放性地固定在檢棒接觸 端與捕獲區之間’取代與檢棒分開。當檢棒接觸端與樣品 溶液接觸導致樣品溶液經由毛細作用移行至捕獲區，偵測 探針釋放到樣品溶液中以至釋放的偵測探針能雜交到已移 行至捕獲區之樣品溶液中之目標核酸。 此分析之進一步變化中，偵測探針可與樣品溶液分開並 與檢棒捕獲區接觸。此項通常進行於檢棒接觸端與樣品溶 液接觸之後。偵測探針可直接接觸捕獲區，或偵測探針在 分開之探針溶液中接觸檢棒接觸端導致樣品溶液經由毛細 作用移行至捕獲區。 2 )提供一種檢棒，含有層析片具有接觸端及捕獲部分固 定在遠離接觸端之捕獲區上，捕獲部分能結合已雜交到目 標核酸之捕獲探針。捕獲探針在捕獲探針能雜交到目標核 酸之條件下接觸樣品溶液。樣品溶液與檢棒接觸端接觸， 使得樣品溶液經由毛細作用移行至捕獲區，藉此使目標核 酸及捕獲探針與樣品溶液移行至捕獲區，並經由捕獲部分 結合捕獲探針將目標核酸捕獲在捕獲區。目標核酸可使用 如分析（1)所述之偵測探針偵測。偵測探針可與捕獲探針 -11- 1286159 五、發明說明（1〇) 一起或分開加入樣品溶液（以任何順序）。另外，偵測探針 可釋放性地固定在檢棒接觸端與捕獲區之間，或如分析（i) 所述分開接觸捕獲區。 分析（2 )之變化中，取代捕獲探針與樣品溶液混合，而 改將捕獲探針可釋放性地固定在檢棒接觸端與捕獲區之間 。當檢棒接觸端與樣品溶液接觸導致樣品溶液經由毛細作 用移行至捕獲區，捕獲探針釋放到樣品溶液中以至釋放的 捕獲探針能雜交到已移行至捕獲區之樣品溶液中之目標核 酸。目標核酸可被偵測，因爲使用之偵測探針可接觸樣品 溶液，可釋放性地固定在檢棒接觸端與捕獲區之間，或分 開接觸捕獲區。 分析（2)之進一步變化中，捕獲探針接觸捕獲區之前（或 例外地同時），樣品溶液經由毛細作用到達捕獲區。如此 可使捕獲探針經由捕獲部分結合在捕獲區以至目標核酸被 捕獲。捕獲探針可在分開之捕獲探針溶液中，經由直接置 於捕獲區而分開接觸捕獲區，或經由捕獲探針溶液接觸檢 棒接觸端導致捕獲探針經由毛細作用移行至捕獲區。連續 的檢棒接觸端與樣品溶液接觸將使目標核酸經由毛細作用 到達捕獲區而被捕獲。再次的，目標核酸可被偵測，因爲 使用之偵測探針可接觸樣品溶液，可釋放性地固定在檢棒 接觸端與捕獲區之間，或分開接觸捕獲區。如有變化的在 分析（2 )中使用偵測探針，目標核酸可直接於樣品溶液中 標識，例如標識以共價附著於目標核酸。此項之達成可經 -12- 1286159 五、發明說明（11 ) 由標識前驅物（p r e c u r s 〇 r 1 a b e 1 )與樣品溶液接觸，並在 標識可共價附著於目標核酸之條件下培育標識前驅物與樣 品溶液。 分析（2)之捕獲部分可爲能雜交到捕獲探針之一般捕獲 探針，或捕獲部分可經由非鹼基配對之交互作用結合到捕 獲探針。例如當捕獲探針含有一或以上捕獲配位體時，捕 獲部分爲捕獲配位體結合部分。 當檢棒分析使用一種以上能雜交到目標核酸之探針時， 較佳爲全部探針加到樣品溶液且於單一步驟中進行雜交作 用。如此簡化分析使其更容易且快速執行。已發現使用單 一步驟雜交分析偵測目標核酸的敏感度約相同於雜交作用 於多步驟進行之偵測作用的敏感度。多步驟雜交作用之進 行可經由不同探針於樣品溶液中對目標核酸之連續雜交， 或經由檢棒接觸各含有不同探針之不同溶液。通常多步驟 雜交作用之後項方法在接觸每一不同探針溶液之間要淸洗 檢棒。然而某些情況中多步驟雜交作用較佳，需知更簡單 及更快速形式之單一步驟雜交作用通常較佳。 最佳爲樣品溶液具有適當的組成物使雜交反應進行於單 一雜交步驟中，也使其進行非鹼基配對之交互作用（例如 偵測配位體及偵測配位體結合部分之間，以及捕獲配位體 及捕獲配位體結合部分之間）並經由毛細作用將含有目標 核酸及一種或多種已雜交探針及（選擇地）配位體結合部分 之複合物在層析片上移傳送。使用如此樣品溶液時’需知 -13- 1286159 五、發明說明（12) 樣品溶液直接與檢棒接觸端接觸之前雜交反應可於單一步 驟中進行’以及可進行任何配位體—配位體結合部分之交 互作用（不需第一次稀釋或變化樣品溶液）。假如一旦需要 樣品溶液移行至捕獲區時，配位體-配位體結合部分之交 互作用能額外或替代地在檢棒進行。藉此促進目標核酸之 簡單且快速的檢棒偵測。 我們發現：此等結果是以含有標準雜交緩衝液（例如 SSPE緩衝液或三鹽緩衝液（Tris buffer))與鹽，淸潔劑及 阻礙性蛋白質（例如BSA或奶粉）之樣品溶液完成之。使用 此種分析之目標核酸偵測之敏感度，已知約與其他檢棒分 析法相當。 根據本發明也提供一種探針，含有序列互補於目標核 酸的序列，藉此使探針能雜交到樣品溶液中的目標核酸 ，以及含有序列不互補於目標核酸的序列，其中非互補 序列位於探針序列之一端，以及當探針雜交到樣品溶液 中之目標核酸時，非互補序列不會經由樣品溶液之成分 結合。 探針以核酸探針爲佳。然而探針可爲核酸類似物，例 如蛋白質核酸（PNA) ° 非互補序列較佳至少三個核苷酸長度’更佳至少六個 核苷酸長度。 互補序列較佳10-1 00核苷酸長度’更佳20 - 3 5核苷酸 長度。 -14- 1286159 五、發明說明（13) 非互補序列較佳位於探針序列的兩端。 較佳爲標識或配位體連接到探針。較佳爲標識或配位體 以共價連接到最遠離互補序列之非互補序列端。 根據本發明也提供使用本發明探針於檢棒分析中測試樣 品溶液中目標核酸之存在。 根據本發明進一步提供使用本發明之檢棒或套組於檢棒 分析中測試樣品溶液中目標核酸之存在。 文中使用之“檢棒分析”一詞意指使用檢棒之任何分析 ，其中樣品溶液與檢棒接觸，經由毛細作用引起樣品溶液 移動到檢棒捕獲區，藉此使樣品溶液中的目標核酸在捕獲 區被捕獲並偵測，目標核酸的捕獲及/或偵測包括捕獲/偵 測經雜交到目標核酸。 茲以僅作爲參考用之實施例及隨文圖式說明本發明具體 實施例，其中： 第1圖爲適用作爲本發明捕獲探針間隔子或偵測探針間 隔子成分之非蛋白質成分的化學結構； 第2圖爲使用本發明具體實施例偵測砂眼披衣菌 (Chlamydia trachomatis)巨標核懷’， 第3圖爲實施例1之實驗構成； 第4圖爲實施例2之實驗構成； 第5圖爲實施例3之實驗構成； 第6圖爲實施例4之實驗構成； 第7圖爲實施例6之實驗構成； -15- 1286159 五、發明說明（14) 第8圖爲實施例7之實驗構成；以及 第9圖爲實施例中所用之偵測探針的結構。 實施例係關於砂眼披衣菌（07CT )) 隱性質體DNA之DNA片段的偵測。CT是最常見之性行爲傳 播引起的疾病之一。CT感染能引起不孕，且於懷孕期間乃 至出生可造成自發性流產或產後子宮內膜炎。在新生兒， CT感染能引起失明及慢性呼吸道疾病。接近1 〇%受感染男 性及高達70%受感染女性未表現CT感染之症狀。因此，CT 感染之正確診斷是重要的，以至能開始此疾病之早期治療 〇 本發明具體實施例中，檢棒1 0是用於測試樣品溶液1 4 中是否有單股或雙股CT目標核酸1 2 (參照第2圖）。檢棒 1 0含有一種硝基纖維片1 6，具有接觸端1 8用於接觸樣品 溶液1 4，以及一種捕獲探針20固定在硝基纖維片1 6遠離 接觸端18之捕獲區22。一種抗-生物素抗體-染料結合物 24 (或實施例5之抗-螢光素抗體-染料結合物）可釋放性 地固定在硝基纖維層析片接觸端1 8與捕獲區22間之結合 區26。捕獲探針20能雜交到目標核酸1 2之一股（第一股） 的第一區域。 樣品溶液1 4製備自加入1 m 1尿液與砂眼披衣菌菌體， 然後以15K r pm旋轉尿液30分鐘。小片（pellet)再懸浮 於1 00 // 1標準雜交緩衝液（包括阻斷劑例如酪蛋白或BSA ) 。然後加入能雜交到目標核酸之偵測探針28(與幫手探針 -16- 1286159 五、發明說明（15) (helper probe)—起，其能雜交到目標核酸鄰近區域，若 使用偵測探針及/或捕獲探針可以辨認之）。偵測探針28 是連接生物素（或實施例5之螢光素）（使用技藝熟知方法） 。然後樣品溶液1 4加熱至1 〇〇 t： 7分鐘然後冷卻。 然後檢棒1 0之接觸端1 8接觸樣品溶液1 4。樣品溶液 1 4與雜交到偵測探針之任何目標核酸1 2經由毛細作用 在檢棒10往上移行。當樣品溶液14通過結合區26時流 動抗-生物素抗體-染料結合物24。之後釋放之抗-生物素 抗體-染料結合物24可結合到已雜交目標核酸12之連接 生物素的偵測探針2 8。 然後抗-生物素抗體-染料結合物24、偵測探針28及目 標核酸1 2間形成之複合物在檢棒1 〇往上移行到捕獲區22 ’於此處複合物的目標核酸能雜交到固定的捕獲探針20。 捕獲探針20因固定在捕獲區22無法因樣品溶液14移行 通過捕獲區22而流動。因此，結合到捕獲探針之複合物 保留在捕獲區，並能以存在於捕獲區之抗-生物素抗體-染 料結合物的染料來偵測。 假設樣品溶液沒有CT目標核酸，則偵測探針28不會在 捕獲區22被捕獲，且捕獲區不會看到染料。假設樣品溶 液中有CT目標核酸，但捕獲區捕獲不足數量的目標核酸 時，將無法偵測樣品溶液中目標核酸的存在。 現已發現：若目標核酸的捕獲探針雜交區域與偵測探針 雜交區域間之距離少於2 6核苷酸時會降低目標核酸的偵 -17- 12861591286159 V. DESCRIPTION OF THE INVENTION (1) The present invention relates to enhanced nucleic acid detection of a test stick (d i p s t i c k s ). The probe of the present invention is used to detect the presence of a target nucleic acid in a sample solution, for example, to identify whether a patient is suffering from a disease-causing microorganism such as Chlamydia trachomatis (Chlamydia trachomatis). Some of the customary tests used to detect the presence of a target nucleic acid in a sample solution rely on the amplification of the target nucleic acid using a polymerase chain reaction (PCR). This reaction can detect small amounts of target nucleic acid, but it takes hours before the results are obtained, but it is a significant disadvantage because it is usually desirable to get results as quickly as possible, such as keeping the patient's waiting time to a minimum. A further disadvantage of these methods is the need for expensive expert techniques for the reaction and the high cost of the reagents. In contrast, the bar detects unmagnified target nucleic acids without any expert technique and results faster than PCR-based methods. Patients can be treated in the same visit, so it is especially beneficial for patients who do not like or can not return to the clinic. In a typical conventional test bar described in US 5,310,650, a single-strand DNA capture probe (c a p t r r e p r 〇 b e ) is immobilized on a nitrocellulose filter paper located away from the capture zone of the filter, the end of the paper (contact end). A portion of the sequence of the capture probe is complementary to the sequence of the first region of the target nucleic acid to be tested. The labeled single-strand DNA detection probe (d e t e c t i ο n p r 〇 b e ) is immobilized on a nitrocellulose filter paper located in the probe zone between the filter paper capture zone and the contact end. The detection probe contains a sequence that is complementary to the sequence of the second region of the target nucleic acid (different from the first region). When the target DNA is detected in the sample solution containing the target DNA, the contact end of the nitrocellulose filter paper is brought into contact with the sample solution. The sample solution is moved over the filter paper and passed through the probe zone and the capture zone via capillary 1286159. As the sample solution passes through the probe zone, the detection probe is moved and causes it to rise toward the capture zone with the sample solution. The moving detection probe can then hybridize to a second region of any target DNA present in the sample solution. When the hybridized detection probe and the target DNA reach the capture zone, the first region of the target DNA can hybridize to the immobilized capture probe. Thereby a tertiary complex is formed between the target nucleic acid, the capture probe and the labeled detection probe. Capture area The presence of the marker indicates the presence of the target DNA in the sample solution. As for the second type of conventional test stick, the labeled DNA detecting probe is not fixed on the nitrocellulose filter paper. Instead, the detection probe is added under conditions that allow the detection probe to hybridize to any target nucleic acid in the sample solution. The contact end of the nitrocellulose filter paper is then contacted with the sample solution, and the target nucleic acid hybridized to the detection probe when the sample solution is moved up the probe can be captured in the capture zone via the capture probe. However, it has been found that the sensitivity of nucleic acid detection of a conventional test stick is low. If the target nucleic acid is double-stranded, the sensitivity of nucleic acid detection is extremely low. Therefore, it is sometimes possible that the target nucleic acid present in the sample solution is not detected. It is therefore desirable to improve the sensitivity of nucleic acid detection by the probe. According to a first aspect of the present invention, there is provided a test bar for testing the presence of a target nucleic acid in a sample solution, the test bar comprising: a chromatography plate having a contact end for contacting the sample solution; and a capture probe fixed to the capture from the contact end In the region, the capture probe can hybridize to the target nucleic acid or the hook capture probe that has bound the target nucleic acid -4- 1286159. (hook c ap ture probe), wherein one or both ends of the capture probe Connecting one or more nucleotides, preferably at least three nucleotides, which do not hybridize to the target nucleic acid when the capture probe hybridizes to the giant nucleic acid, or when the capture probe hybridizes to the hook capture probe Will hybridize to the hook capture probe. According to a first aspect of the present invention, there is also provided a kit for testing the presence of a giant nucleic acid in a sample solution comprising a test probe according to the present invention, and a detection probe capable of hybridizing to a target nucleic acid, thereby utilizing detection Needle Detection of Target Nucleic Acids The term "chromatography" as used herein refers to any permeable sheet material that can deliver a solution by capillary action. The detection probe can be releasably fixed to the probe region at the contact end of the probe and the capture interval. One or more nucleotides of the detection probe can be connected to one or more nucleotides, preferably at least three a nucleotide which does not hybridize to a target nucleic acid when the detection probe hybridizes to the target nucleic acid. According to the second part of the present invention, a test rod is also provided for testing the presence of a giant nucleic acid in a sample solution, which comprises: The chromatogram has a contact end for: contacting the sample solution; a capture moiement (capture mo i ety) is fixed directly or indirectly to the capture portion of the capture region remote from the contact end; binding the target nucleic acid; and detecting a probe Releasably immobilized in the probe region of the contact end and the capture interval y. The detection probe can hybridize to the target nucleic acid, thereby detecting the target nucleic acid by using the detection probe, wherein one or two of the probe-5-needle are detected. The end can be connected or 1286159. V. Description of the invention (4) More nucleotides, preferably at least three Nucleotide, which detects when the probe is hybridized to a labeled nucleic acid will not hybridize giant giant-labeled nucleic acid. According to the present invention there is further provided a kit for testing the presence of a giant nucleic acid in a sample solution comprising: a rod according to the second part of the invention, wherein the capture moiety is capable of binding to a capture ligand (which is connected To a capture probe that has bound a target nucleic acid;; a capture probe capable of hybridizing to a giant nucleic acid or a target nucleic acid, and a capture probe attached to the capture ligand One or more nucleotides, preferably at least two nucleotides, which are capable of binding to one or both ends of the capture probe via the capture moiety, do not hybridize to the giant marker when the capture probe hybridizes to the giant nucleic acid The nucleic acid, or when the capture probe hybridizes to the hook capture probe, does not hybridize to the hook capture probe. Also provided in accordance with the invention is a kit for testing the presence of a target nucleic acid in a sample solution comprising: i) a test strip containing a chromatographic strip having a contact end for contacting the sample solution and a capture probe capable of hybridizing to the target The nucleic acid or the hook that has been bound to the target nucleic acid captures the probe, and the capture probe is immobilized on the capture sheet away from the contact capture 13⁄4 and the detection probe can hybridize to the 1 target core, thereby Detecting a target nucleic acid using a detection probe, wherein one or both ends of the detection probe can be linked to -* or more nucleotides, preferably at least three nucleotides, which are hybridized to the target when the detection probe is hybridized Nucleic acids do not interfere with the parent; • 6 - nucleic acid. 1286159 V. INSTRUCTION DESCRIPTION (5) According to the present invention there is also provided a kit for testing the presence of a target nucleic acid in a sample solution, comprising: i) a test strip comprising a chromatography strip having a contact end for contacting a sample solution, and A capture moiety is immobilized on the capture region of the chromatogram away from the contact end, and the capture moiety can directly or indirectly bind the target nucleic acid; ii) a capture probe can hybridize to the target nucleic acid or a hook capture probe that has bound the target nucleic acid, wherein capture The probe is coupled to capture the ligand to be capable of binding via the capture moiety; and iii) a detection probe is capable of hybridizing to the target nucleic acid, wherein one or both ends of the detection probe are ligated to one or more nucleotides, preferably At least three nucleotides that do not hybridize to the target nucleic acid when the detection probe hybridizes to the target nucleic acid. According to the present invention there is also provided a kit for testing the presence of a target nucleic acid in a sample solution comprising: i) a test strip comprising a chromatographic strip having a contact end for contacting the sample solution, and a capture portion immobilized on the chromatogram a capture zone remote from the contact end; 1 1) a capture probe capable of hybridizing to a target nucleic acid or a hook capture probe that has bound a target nucleic acid, wherein the capture probe is coupled to the capture ligand to be capable of binding via the capture moiety, and capture probe One or both ends of the needle may be linked to one or more nucleotides, preferably at least three nucleotides, which do not hybridize to the target nucleic acid when the capture probe hybridizes to the target nucleic acid; and iii) a detection probe The target nucleic acid can be hybridized to detect the target nucleic acid using a detection probe. The detection rod and the set of the invention can be used for testing the presence of the target nucleic acid in the sample solution. 1286159 5. The detection probe capable of hybridizing to the target nucleic acid in the method of the invention (6) can hybridize to the detection of the probe nucleic acid Incubate with the sample solution under conditions. The contact of the test bar touches the sample solution to make the sample solution move upward through the capillary to the test rod. 0 Then the target nucleic acid hybridized to the detection probe in the sample solution can be captured by the capture T& The presence of the target nucleic acid in the sample solution can be indicated by the detection probe in the capture zone. However, the detection probe is releasably immobilized on the probe, for example, in the probe region at the contact end of the chromatogram and the capture interval, and the contact end of the probe is brought into contact with the sample solution until the sample solution moves up through the capillary to the inspection rod. . The detection probe flows when the sample solution passes through the probe probe region, so that the detection probe can hybridize to the giant nucleic acid in the sample solution and migrate to the capture region with the target nucleic acid, and the sample solution passes through the n' In the JH, m capture region, the target nucleic acid hybridized to the detection probe is captured via the capture probe. The presence of the target nucleic acid in the sample solution can be indicated by the detection probe in the capture region. The capture moiety can be directly or indirectly bound to the macronucleic acid by interaction of base pairing or non-base pairing. For example, the capture portion can contain a capture probe that can be directly hybridized to the target nucleic acid. In addition, the capture moiety may contain a capture probe> which can hybridize directly to the hook capture probe that has bound the target nucleic acid. The capture moiety may bind to the capture ligand (a capture probe that has been ligated to the bound g nucleic acid) Thereby, the capture moiety is indirectly bound to the target nucleic acid. For example, the j capture moiety may be an antibody or an antibody fragment. If the capture moiety is attached to the capture ligand, the capture probe may be attached to a link-8-capture 1286159. 5, invention note (7) ligand capture probe spacer, and capture coordination The body is spaced from the capture probe. The hook capture probe can be added to the sample solution to bind to the target nucleic acid in the sample solution and captured by the capture probe as the sample solution moves upward through the capillary via the capillary. The capture probe, hook capture probe and detection probe can each contain at least one nucleic acid or nucleic acid analog. Where the probe herein contains more than one nucleic acid or nucleic acid analog, it is preferred to hybridize together. The detection probe can be coupled to the label to enable direct detection of the target nucleic acid in the capture zone. In addition, the detection probe can be linked to the detection ligand to detect the target nucleic acid indirectly by detecting the binding portion of the ligand. Suitable labels include, for example, textile dyes, metal sols such as gold colloids, and colored particles such as colored latex particles. These markers can be directly connected to the detection probe or, if the detection probe is connected to a detection ligand, directly connected to the detection ligand binding portion. Suitable for capturing or detecting ligands, for example including biotin (for example, capture or detect via avidin antibody, avidin, avidin (str ep tavidi η ), or a derivative thereof) Luciferin (eg, captured or detected via an anti-luciferin antibody) and 2,4-dinitrophenol (DNP) (eg, captured or detected via an anti-DNP antibody). The detection probe can contain a general detection probe that hybridizes to a hook detection probe that has bound the target nucleic acid. A general detection probe can be connected to the identification or detection ligand to detect the detection probe. It is to be understood that the kit of the present invention may further comprise any reagent required for the detection of the sample solution using the kit 1286159. For example, the kit of the invention may comprise a detection probe linked to the detection ligand, which may further comprise a detection ligand binding moiety. 侦测 The detection ligand binding moiety may comprise an antibody or antibody fragment, or a non-antibody. For example, if the detection ligand contains biotin, the detection ligand binding moiety may contain an anti-biotin antibody, streptavidin, avidin or a derivative thereof which retains biotin-binding activity. Preferably, the detection partner binding moiety is identified, thereby enabling indirect detection of the target nucleic acid by using the detection probe and detecting the binding moiety of the ligand. It is to be noted that the present invention relates to the use of a detection probe and/or a capture probe for connecting one or more "unpaired" nucleotides, and the detection probe or capture probe can be attached to a test stick or a target. Nucleic acid detection methods and sample solution incubations. The use of nucleobase-containing unpaired nucleotides according to the present invention is considered to be particularly effective, depending on the nucleotide or nucleobase energy and formation of the capture probe or detection. The base between the probe and the target nucleic acid forms a stacking interaction. The formation of a stacking interaction is believed to enhance hybridization of the capture probe or detection probe to the target nucleic acid, thereby improving the capture or detection efficiency of the target nucleic acid in the rod capture zone. Where appropriate, the test rods and kits of the present invention can be used for the presence of target nucleic acids in a test sample solution for the following types of assays: 1) Providing a test strip containing a chromatographic sheet having a contact end and a capture probe fixed in contact with the contact At the capture region of the end, the capture probe hybridizes to the target nucleic acid. A detection probe that can hybridize to a target nucleic acid under the condition that the detection probe can hybridize to the target nucleic acid. -10- 1286159 V. Description of the invention (9) Contact the sample solution. The sample solution is contacted with the contact end of the rod so that the sample solution migrates to the capture zone via capillary action, whereby the target nucleic acid and the detection probe and the sample solution are transferred to the capture zone, and the target nucleic acid is captured in the capture zone. The presence of the target nucleic acid in the sample solution can be indicated by the presence of a detection probe in the capture zone. In this variation of the assay, the detection probe is releasably secured between the contact end of the rod and the capture zone' instead of being separated from the detector. When the contact end of the rod contacts the sample solution, the sample solution migrates to the capture zone via capillary action, and the detection probe is released into the sample solution so that the released detection probe can hybridize to the target solution that has migrated to the sample solution in the capture zone. Nucleic acid. In a further variation of this analysis, the detection probe can be separated from the sample solution and contacted with the rod capture zone. This is usually done after the contact end of the rod contacts the sample solution. The detection probe can be in direct contact with the capture zone, or the detection probe contacts the contact end of the probe in a separate probe solution causing the sample solution to migrate via capillary action to the capture zone. 2) A test rod comprising a chromatographic sheet having a contact end and a capture portion affixed to a capture region remote from the contact end, the capture portion being capable of binding to a capture probe that has hybridized to the target nucleic acid. The capture probe contacts the sample solution under conditions in which the capture probe can hybridize to the target nucleic acid. The sample solution is in contact with the contact end of the rod, so that the sample solution migrates to the capture zone via capillary action, thereby moving the target nucleic acid and the capture probe and the sample solution to the capture zone, and capturing the target nucleic acid via the capture moiety binding capture probe Capture area. The target nucleic acid can be detected using a detection probe as described in analysis (1). The detection probe can be added to the sample solution (in any order) together with or separately from the capture probe -11- 1286159 V. Instructions for the Invention (1〇). Alternatively, the detection probe is releasably secured between the contact end of the rod and the capture zone or separately from the capture zone as described in analysis (i). In the variation of analysis (2), the replacement capture probe is mixed with the sample solution, and the capture probe is releasably fixed between the contact end of the probe and the capture zone. When the contact end of the rod contacts the sample solution causing the sample solution to migrate to the capture zone via capillary action, the capture probe is released into the sample solution such that the released capture probe can hybridize to the target nucleic acid in the sample solution that has migrated to the capture zone. The target nucleic acid can be detected because the detection probe used can be contacted with the sample solution, releasably immobilized between the contact end of the probe and the capture zone, or separated from the capture capture zone. In a further variation of analysis (2), before the capture probe contacts the capture zone (or alternatively, at the same time), the sample solution reaches the capture zone via capillary action. Thus, the capture probe can be bound to the capture zone via the capture moiety until the target nucleic acid is captured. The capture probe can be contacted separately in the capture probe solution by placing it directly in the capture zone, or contacting the probe contact via the capture probe solution to cause the capture probe to migrate via capillary action to the capture zone. Contact of the continuous rod contact end with the sample solution will cause the target nucleic acid to be captured via capillary action to the capture zone. Again, the target nucleic acid can be detected because the detection probe used can be contacted with the sample solution, releasably attached between the contact end of the probe and the capture zone, or separately from the capture zone. If a detection probe is used in analysis (2), the target nucleic acid can be identified directly in the sample solution, e.g., the label is covalently attached to the target nucleic acid. This can be achieved by -12- 1286159. V. Inventive Note (11) The precursor of the label (precurs 〇r 1 abe 1 ) is contacted with the sample solution, and the label precursor is cultivated under the condition that the label can be covalently attached to the target nucleic acid. And sample solution. The capture moiety of assay (2) can be a general capture probe that hybridizes to the capture probe, or the capture moiety can be coupled to the capture probe via non-base pairing interactions. For example, when the capture probe contains one or more capture ligands, the capture moiety is the capture ligand binding moiety. When the rod assay uses more than one probe capable of hybridizing to the target nucleic acid, preferably all probes are added to the sample solution and hybridized in a single step. This simplifies the analysis to make it easier and faster to perform. It has been found that the sensitivity of detecting a target nucleic acid using a single-step hybridization assay is about the same as the sensitivity of the hybridization to a multi-step detection. Multi-step hybridization can be carried out by successive hybridization of the target nucleic acid in the sample solution via different probes or by contacting different solutions containing different probes via a probe. Typically, the multi-step hybridization method will wash the rod between each different probe solution. However, in some cases multi-step hybridization is preferred, and it is generally preferred that single-step hybridization in a simpler and faster form is generally preferred. Optimally having a suitable composition for the sample solution to allow the hybridization reaction to proceed in a single hybridization step, also allowing for non-base pairing interactions (eg, detecting ligands and detecting ligand binding moieties, and The capture ligand and the capture ligand binding moiety are ligated and the complex containing the target nucleic acid and one or more of the hybridized probes and (optionally) the ligand binding moiety is transported over the chromatogram via capillary action. When using such a sample solution, 'Required-13- 1286159 V. Inventive Note (12) The hybridization reaction can be carried out in a single step before the sample solution is directly contacted with the contact end of the rod' and any ligand-ligand binding can be performed. Partial interaction (no need to first dilute or change the sample solution). If the sample solution is required to migrate to the capture zone, the interaction of the ligand-ligand binding moiety can additionally or alternatively be performed on the rod. This facilitates simple and rapid detection of the target nucleic acid. We have found that these results are accomplished with sample solutions containing standard hybridization buffers (such as SSPE buffer or Tris buffer) with salts, detergents, and barrier proteins such as BSA or milk powder. The sensitivity of target nucleic acid detection using such assays is known to be comparable to other rod assays. According to the present invention there is also provided a probe comprising a sequence complementary to a target nucleic acid, whereby the probe is capable of hybridizing to a target nucleic acid in a sample solution, and a sequence comprising a sequence not complementary to the target nucleic acid, wherein the non-complementary sequence is located One end of the needle sequence, and when the probe hybridizes to the target nucleic acid in the sample solution, the non-complementary sequence does not bind via the components of the sample solution. The probe is preferably a nucleic acid probe. However, the probe may be a nucleic acid analog, such as a protein nucleic acid (PNA) ° non-complementary sequence preferably at least three nucleotides in length more preferably at least six nucleotides in length. The complementary sequence is preferably from 10 to 10 nucleotides in length, more preferably from 20 to 3 5 nucleotides in length. -14- 1286159 V. INSTRUCTIONS (13) Non-complementary sequences are preferably located at both ends of the probe sequence. Preferably the label or ligand is attached to the probe. Preferably, the label or ligand is covalently linked to the non-complementary sequence end furthest from the complementary sequence. The presence of a target nucleic acid in a test sample solution for use in a rod assay using the probe of the present invention is also provided in accordance with the present invention. According to the present invention there is further provided the presence of a target nucleic acid in a test sample solution for use in a rod assay using a test rod or kit of the present invention. As used herein, the term "checking bar" means any analysis using a test bar in which a sample solution is contacted with a test bar to cause the sample solution to move to the trap capture zone via capillary action, thereby allowing the target nucleic acid in the sample solution to be The capture zone is captured and detected, and capture and/or detection of the target nucleic acid includes capture/detection of hybridization to the target nucleic acid. DETAILED DESCRIPTION OF THE INVENTION The present invention is described by way of example only and with reference to the accompanying drawings, in which: FIG. 1 is a chemistry which is applied as a non-protein component of the capture probe spacer or the probe probe spacer component of the present invention. Fig. 2 is a diagram showing the use of the present invention to detect the Chlamydia trachomatis giant nucleus, Fig. 3 is the experimental composition of the embodiment 1, and Fig. 4 is the experimental composition of the second embodiment; Fig. 5 is an experimental composition of the embodiment 3; Fig. 6 is an experimental constitution of the embodiment 4; Fig. 7 is an experimental composition of the embodiment 6; -15 - 1286159 5. Description of the invention (14) Fig. 8 is an embodiment The experimental composition of 7; and Fig. 9 is the structure of the detection probe used in the examples. The examples relate to the detection of DNA fragments of cryptic plastid DNA by Chlamydia trachomatis (07CT). CT is one of the most common diseases caused by sexual transmission. CT infection can cause infertility and can cause spontaneous abortion or postpartum endometritis during pregnancy or even birth. In newborns, CT infection can cause blindness and chronic respiratory diseases. Nearly 1% of infected men and up to 70% of infected women did not show symptoms of CT infection. Therefore, the correct diagnosis of CT infection is important so that early treatment of the disease can be initiated. In a specific embodiment of the invention, the test rod 10 is used to test whether there is a single or double-strand CT target nucleic acid in the sample solution 14 1 2 (Refer to Figure 2). The rod 10 contains a nitrocellulose sheet 16 having a contact end 18 for contacting the sample solution 14 and a capture probe 20 fixed to the capture zone 22 of the nitrocellulose sheet 16 away from the contact end 18. An anti-biotin antibody-dye conjugate 24 (or the anti-luciferin antibody-dye conjugate of Example 5) is releasably immobilized between the contact end 18 of the nitrocellulose chromatography sheet and the capture zone 22. Bonding zone 26. The capture probe 20 is capable of hybridizing to a first region of one of the target nucleic acids 12 (first strand). The sample solution 14 was prepared by adding 1 ml of urine to the bacterium of Chlamydia trachomatis, and then rotating the urine at 15 K r pm for 30 minutes. The pellet is resuspended in 1 000 // 1 standard hybridization buffer (including blockers such as casein or BSA). Then, a detection probe 28 capable of hybridizing to the target nucleic acid is added (with the helper probe-16-1286159, the helper probe, which can hybridize to the vicinity of the target nucleic acid, if detection is used The needle and / or capture probe can be identified). Detection probe 28 is a biotin (or luciferin of Example 5) (using methods well known in the art). The sample solution 14 was then heated to 1 〇〇 t: 7 minutes and then cooled. The contact end 18 of the rod 10 is then contacted with the sample solution 14 . The sample solution 14 and any target nucleic acid 12 that hybridizes to the detection probe migrates upward through the rod 10 via capillary action. The anti-biotin antibody-dye conjugate 24 is flowed as the sample solution 14 passes through the binding zone 26. The anti-biotin antibody-dye conjugate 24 which is then released can bind to the biotin-detecting probe 28 which has hybridized to the target nucleic acid 12. Then, the complex formed between the anti-biotin antibody-dye conjugate 24, the detection probe 28 and the target nucleic acid 12 moves up to the capture zone 22' where the target nucleic acid of the complex can hybridize. To the fixed capture probe 20. The capture probe 20 cannot flow due to migration of the sample solution 14 through the capture zone 22 due to immobilization in the capture zone 22. Thus, the complex bound to the capture probe remains in the capture zone and can be detected by the dye present in the capture zone of the anti-biotin antibody-dye conjugate. Assuming that the sample solution does not have a CT target nucleic acid, the detection probe 28 will not be captured in the capture zone 22 and the dye will not be visible in the capture zone. Assuming that there is a CT target nucleic acid in the sample solution, but the capture region captures an insufficient number of target nucleic acids, the presence of the target nucleic acid in the sample solution cannot be detected. It has been found that if the distance between the capture probe hybridization region of the target nucleic acid and the detection probe hybridization region is less than 26 nucleotides, the target nucleic acid will be reduced. -17-1286159
五、發明說明（16) 測敏感度。因此，較佳爲這些區域間之距離爲至少26核 苷酸，且較佳爲至少200核苷酸。 上述目標核酸的捕獲是指以下列實施例中直接探針捕獲 。實施例中目標核酸的偵測強度以0到5等級記錄，5代 表偵測強度最強，以及0代表沒有偵測強度。 下列實施例使用之探針序列如下所示： SEQ ID NO : 1 : 5， TGC AAC TCT TGG TGG TAG ACT TTG C SEQ ID NO : 2 : 5, GCG CAC AGA CGA TCT ATT TTT TGC A SEQ ID NO : 3 : 5， CGG GCG ATT TGC CTT AAC CCC ACC A SEQ ID NO : 4 : 5， CCA AGC TTA AGA CTT CAG AGG AGC G SEQ ID N〇：5 : 5， CAT GCG TTT CCA ATA GGA TTC TTG G SEQ ID NO : 6 ·· 5, CAC AGT CAG AAA TTG GAG TGC TGG C SEQ ID NO : 7 : 5， CTT GCT GCT CGA ACT TGT TTA GTA C SEQ ID NO : 8 : 5, AGA AGT CTT GGC AGA GGA AAC TTT T SEQ ID NO : 9 : 5， CTA GAA TTA GAT TAT GAT TTA AAA GGG SEQ ID NO ： 10 ： 59 TTC ATA TCC AAG GAC AAT AGA CCA A SEQ ID NO : 11 : 5’ TGA TCT ACA AGT ATG TTT GTT GAG T SEQ ID NO : 12 : 5， TGC ATA ATA ACT TCG AAT AAG GAG AAG SEQ ID NO : 13 : 5， TCC CTC GTG ΑΤΑ TAA CCT ATC CG SEQ ID NO : 14 : 5， CAG GTT GTT AAC AGG ATA GCA CGC SEQ ID NO : 15 : 5， CTC GTT CCG AAA TAG AAA ATC GCA SEQ ID NO : 16 : 5’ GGT AAA GCT CTG ATA TTT GAA GAC SEQ ID NO : 17 : 5， CTG AGG CAG CTT GCT AAT TAT GAG T -18- 1286159 五、發明說明（17) 下述實施例中使用之偵測探針的結構圖式於第9圖。 實施例1 實驗構成 捕獲格式：固定在檢棒之直接探針捕獲（cp) Seq ID No 14 ； 偵測探針（dp):以生物素直接連接到5’-端，或經由3 核苷酸（N3)，6核苷酸（N6)，S，或SS間隔子連接Seq ID No 13或 Seq ID No 15，或以生物素直接連接到SEQ ID No 13之3’ -端。每一各1012複本。 偵測格式：抗-生物素抗體-染料結合物； 目標DNA: 73或76核苷酸之單股DNA片段，5xlOu-1 〇1 ^複本。 糸吉果 do Seq ID No 13 目標之複本： 5xl0n 1011 5xl010 101ϋ 偵測探針 訊號強度 dp-B5 3.5 3.0 2.0 1.0 dp - N3 - B5 4.5 3.5 3.0 1.5 dp - Νό - B5 5.0 4.0 3.0 2.0 dp-S-B5， 4.0 3.0 2.0 1.0 dp-SS-B5’ 4.5 3.5 2.5 1.0 3，B-dp 5.0 3.5 3.0 2.0 -19- 1286159 五、發明說明（18) dp Sea ID No 15 目標之複本： 5xl0n 1011 5x10 偵測探針 訊號強度 dp-B5 3.5 2.0 1.0 dp - N3 4.0 3.0 2.0 dp - - B5 4.5 3.0 2.0 dp-S-B5’ 4.0 2.5 1.5 dp-SS-B5’ 4.0 2.5 1.5 這些結果顯示： 使用經由N3，N6，S，或SS間隔子將生物素連接到5’-端之偵測探針於目標核酸偵測之敏感度，高於使用生物素 直接連接到5’ -端之偵測探針於目標核酸偵測之敏感度。 N3及N6間隔子比S及SS間隔子好。 較佳爲生物素（或其他偵測配位體）連接到偵測探針的一 端。在捕獲區當捕獲探針及偵測探針雜交到目標核酸形成 之複合物中，生物素（或其他偵測配位體）可能在偵測探針 之一端接近目標核酸雜交捕獲探針之區域（內部方位），或 較佳爲在偵測探針之一端遠離目標核酸雜交捕獲探針之區 域（外部方位）。若沒使用間隔子連接偵測配位體與偵測探 針，若偵測配位體在外部方位則目標核酸之偵測敏感度通 常較高。因此，通常選擇偵測配位體在外部方位之偵測探 -20- 1286159 五、發明說明（19) 針。 然而，在此實施例中使用外部方位生物素連接到偵測探 針3 ’ -端之偵測探針的偵測敏感度’相同於以內部方位生 物素經由六核苷酸間隔子連接到偵測探針5 ’ -端之偵測探 針的偵測敏感度。如此則根據本發明使用間隔子時，選擇 偵測探針不必一定需偵測配位體是位於捕獲於捕獲區之複 合物的外部方位。 實施例2 實驗構成 捕獲格式：固定在檢棒膜之直接探針捕獲（cp) Seci ID No 14 ； 偵測探針：經由3核苷酸（N3)，6核苷酸（N6)，S，或SS 間隔子連接生物素到偵測探針（dp) Seci ID No 13、 Seq ID No 15 及 Seq ID No 16 之 5’-端。每一各 l〇12 複本。 偵測格式：抗-生物素抗體-染料結合物； 目標DNA : 214 bp雙股DNA片段，1011 - 101G複本。 -21 - 1286159 五、發明說明（20) 結果 目標之複本： 1011 5x10 丨0 10丨0 偵測探針 訊號jj皇 dpl3-B+dpl5-B+dpl6-B 1.5 1.0 0.0 dpl3-N3-B+dpl5-N3-B+dpl6-N3-B 3.0 2.0 0.5 dpl3-N6-B+dpl5-N6-B+dpl6-N6-B 4.5 3.0 1.0 dpl3-S-B+dpl5-S-B+dpl6-S-B 3.0 2.0 <0.5 dpl3-SS-B+dpl5-SS-B+dpl6-SS-B 3.5 3.0 0.5 dpl3 無標識+dpl5-N6-B+dpl6-N6-B 2.5 1.5 0.5 這些結果顯示： 使用N3，N6，S，或SS間隔子可改良雙股目標核酸的偵 測敏感度超過5倍。 N6及SS間隔子比N3及S間隔子具有較高的偵測敏感度 ，表示間隔子長度對於偵測敏感度的改良很重要° N6間隔子比SS間隔子具有較高的偵測敏感度’儘管事 實上此等間隔子長度相等，表示間隔子物化性質對於偵測 敏感度的改良很重要。 使用僅兩偵測探針各別以N6間隔子連接生物素到5 ’ -端 (#13無標識+(1?15-〜^ + (^16-〜^)(其中生物素位於以 捕獲探針捕獲之複合物的內部方位）的偵測敏感度’大於 使用三偵測探針各別直接連接生物素到5 ’ -端（dp 13- -22- 1286159 五、發明說明（21) B + dpl5-B + dpl6-B)(其中一偵測探針（dpl3-B)的生物素位 於以捕獲探針捕獲之複合物的外部方位）的偵測敏感度。 實施例1及實施例2之結論 目標核酸偵測之敏感度可經由使用間隔子連接偵測配位 體到偵測探針而增加。 較長的間隔子比短的間隔子好。 相同長度但不同物化性質之間隔子對偵測敏感度有不同 的影響。特別是間隔子中非蛋白質成分僅由核苷酸構成者 較佳於間隔子中含有非核苷酸成分者。 此等結果的可能解釋： 1 .核苷酸間隔子改良偵測敏感度可能是經由增強偵測探 針對目標核酸的雜交作用。當偵測探針雜交到目標核酸時 ，這些間隔子的核苷酸不預期會鹼基配對到目標核酸的核 苷酸。這些核苷酸的核鹼基可與鹼基對（形成於偵測探針 雜交到目標核酸時）形成堆積交互作用。這些堆積交互作 用可增強目標核酸與偵測探針間形成之雜交物（hyb I* i d )的 穩定性，藉此增強目標核酸的偵測敏感度。 2.核苷酸間隔子比S及SS間隔子更堅硬。相較於S及 SS間隔子的聚乙二醇基，核苷酸間隔子的核糖環預期可提 供更堅硬性。較大的堅硬性可能增加用與偵測配位體結合 部分交互作用之已連接核苷酸間隔子之偵測配位體的可獲 性。 3 .核苷酸與非核苷酸間隔子之間極性不同，可能導致偵 -23- 1286159 五、發明說明（22 ) 測的敏感度不同。 實施例3 實驗構成 捕獲格式：固定在檢棒之直接探針捕獲（cp) SeQ ID No 10 ； 偵測探針：直接或經由N6，SS，（ds)6，（Sc3)6或 SNJNJ間隔子連接生物素到偵測探針（dp) seq id No 13 之5 ’ -端。每一各1 012複本。 偵測格式：抗-生物素抗體-染料結合物； 幫手探針（helper probes) : SEQ I]) No 5 及 SEQ ID No 6 鄰近 SEQ ID No 10 ; SEQ ID No 1 及 SEq π No 2 鄰近 SEQ ID No 13，1012 複本； 目標DNA : 872 bp雙股DNA片段，： 410^-5^101° 複本 結果 目標之複本： 2xl〇" 5xl〇10 偵測探針 訊號強度 dp-B <1.0 0.0 dp-N6-B 2.0 0.5 dp-SS-B 1.0 0.0 dp-(dS)6-B 1.0 0.0 dp- (SC3)6-B 1.0 0.0 dp-SN3SN3S-B 1.5 0.0 -24- 1286159 五、發明說明（23) 這些結果顯示： 儘管SS ’（dS)6及（sc3 )6間隔子的結構不同與性質之外 ，具有相同長度且在增強目標核酸的偵測敏感度上具有類 似效應。 I間隔子長度相同於SS，（dS)6 &(SC3)6間隔子，然而 N6間隔子在改良目標核酸的偵測敏感度上具有最大效應。 使用N6間隔子比sn3sn3s間隔子（最長的受試間隔子）具 有較大的目標核酸偵測敏感度。此情形之可能解釋爲：S 單體減少或消除間隔子N3成分之核鹼基與鹼基對（形成於 偵測探針與目標核酸之間）間的堆積交互作用。此數據支 持本結論，即間隔子的未配對核鹼基與形成於目標核酸及 偵測探針間之雙體的堆積交互作用在增強目標核酸的偵測 敏感度上很重要。 實施例4 具有不同物化性質及長度之間隔子是以檢棒測試及點墨 分析法評估。缺乏偵測探針雜交到目標核酸時，點墨分析 法能使抗生物素抗體與欲分析之連接抗生物素之偵測探針 的交互作用有效率。5 X 1 08 - 5 X 1 011複本之以不同間隔子連 接抗生物素之偵測探針，點在正價尼龍膜的不同位置，並 對膜UV -交聯。然後培育膜與連接鹼性磷酸酶（能轉換氮藍 四唑/ 5-溴-4-氯-3-吲哚磷酸（NBT/BC IP)產色性受質）之抗 生物素抗體，淸洗，並培育NBT/BCIP產色性受質，然後 觀察膜是否看到任何顏色形成於捕獲區。 -25- 1286159 五、發明說明（24) 檢棒測試之實驗構成 捕獲格式：固定在檢棒之直接探針捕獲（cp)Seq ID No 14 ； 偵測I探針··直接或經由核苷酸或非核苷酸間隔子連接生 物素到偵測探針（dp) Seq ID No 13之 5’ -端。每一各 1012複本。 偵測格式：抗-生物素抗體-染料結合物； 幫手探針：SEQ ID No 2 及 SEQ ID No 3 鄰近 SEQ ID No 14，1〇12 複本； 目標 DNA : 416 bp 雙股 DNA 片段，5xl01G-5xl09 複本。 結果 目標之複本： 2χ10η 5χ101ϋ 偵測探針 訊號強度V. Description of invention (16) Measure sensitivity. Accordingly, it is preferred that the distance between these regions be at least 26 nucleotides, and preferably at least 200 nucleotides. Capture of the above target nucleic acid means capture by direct probe in the following examples. In the examples, the detection intensity of the target nucleic acid is recorded on a scale of 0 to 5, with 5 being the strongest detection intensity and 0 representing no detection intensity. The probe sequences used in the following examples are as follows: SEQ ID NO: 1 : 5, TGC AAC TCT TGG TGG TAG ACT TTG C SEQ ID NO: 2: 5, GCG CAC AGA CGA TCT ATT TTT TGC A SEQ ID NO : 3 : 5, CGG GCG ATT TGC CTT AAC CCC ACC A SEQ ID NO : 4 : 5, CCA AGC TTA AGA CTT CAG AGG AGC G SEQ ID N〇: 5 : 5, CAT GCG TTT CCA ATA GGA TTC TTG G SEQ ID NO : 6 ·· 5, CAC AGT CAG AAA TTG GAG TGC TGG C SEQ ID NO : 7 : 5, CTT GCT GCT CGA ACT TGT TTA GTA C SEQ ID NO : 8 : 5, AGA AGT CTT GGC AGA GGA AAC TTT T SEQ ID NO: 9: 5, CTA GAA TTA GAT TAT GAT TTA AAA GGG SEQ ID NO : 10 : 59 TTC ATA TCC AAG GAC AAT AGA CCA A SEQ ID NO : 11 : 5' TGA TCT ACA AGT ATG TTT GTT GAG T SEQ ID NO: 12: 5, TGC ATA ATA ACT TCG AAT AAG GAG AAG SEQ ID NO: 13 : 5, TCC CTC GTG ΑΤΑ TAA CCT ATC CG SEQ ID NO : 14 : 5, CAG GTT GTT AAC AGG ATA GCA CGC SEQ ID NO : 15 : 5, CTC GTT CCG AAA TAG AAA ATC GCA SEQ ID NO : 16 : 5' GGT AAA GCT CTG ATA TTT GAA GAC SEQ ID NO : 17 : 5, CTG AGG CAG CTT GCT AAT TAT GAG T -18 - 1 286159 V. DESCRIPTION OF THE INVENTION (17) The structural diagram of the detecting probe used in the following examples is shown in Fig. 9. Example 1 Experimental Composition Capture Format: Direct probe capture (cp) fixed to the probe Seq ID No 14; Detection probe (dp): directly linked to the 5'-end with biotin, or via 3 nucleotides (N3), a 6 nucleotide (N6), S, or SS spacer is ligated to Seq ID No 13 or Seq ID No 15, or directly linked to the 3'-end of SEQ ID No 13 with biotin. Each 1012 replica. Detection format: anti-biotin antibody-dye conjugate; target DNA: single strand DNA fragment of 73 or 76 nucleotides, 5xlOu-1 〇 1 ^ copy.糸吉果 do Seq ID No 13 Replica of the target: 5xl0n 1011 5xl010 101ϋ Detection probe signal strength dp-B5 3.5 3.0 2.0 1.0 dp - N3 - B5 4.5 3.5 3.0 1.5 dp - Νό - B5 5.0 4.0 3.0 2.0 dp-S -B5, 4.0 3.0 2.0 1.0 dp-SS-B5' 4.5 3.5 2.5 1.0 3, B-dp 5.0 3.5 3.0 2.0 -19- 1286159 V. Description of invention (18) dp Sea ID No 15 Replica of the target: 5xl0n 1011 5x10 Detect Probe signal strength dp-B5 3.5 2.0 1.0 dp - N3 4.0 3.0 2.0 dp - - B5 4.5 3.0 2.0 dp-S-B5' 4.0 2.5 1.5 dp-SS-B5' 4.0 2.5 1.5 These results show: Use via N3, The N6, S, or SS spacer connects the biotin to the 5'-end detection probe for sensitivity to target nucleic acid detection, higher than the detection probe that is directly attached to the 5'-end using biotin. The sensitivity of nucleic acid detection. The N3 and N6 spacers are better than the S and SS spacers. Preferably, biotin (or other detection ligand) is attached to one end of the detection probe. In the capture zone, when the capture probe and the detection probe hybridize to the complex formed by the target nucleic acid, biotin (or other detection ligand) may be close to the target nucleic acid hybridization capture probe at one end of the detection probe. (internal orientation), or preferably at the end of the detection probe away from the target nucleic acid hybridization capture probe (outside orientation). If the spacer is not detected and the probe is detected, the detection sensitivity of the target nucleic acid is generally high if the ligand is detected in an external orientation. Therefore, it is usually chosen to detect the detection of the ligand in an external orientation. -20- 1286159 V. Inventive Note (19) Needle. However, in this embodiment, the detection sensitivity of the detection probe at the 3 '-end of the detection probe using the external orientation biotin is the same as that of the internal orientation biotin via the hexanucleotide spacer. The detection sensitivity of the detection probe at the 5'-end of the probe is measured. Thus, when a spacer is used in accordance with the present invention, the selection of the detection probe does not necessarily require detection of the ligand being in an external orientation of the complex captured in the capture zone. Example 2 Experimental Composition Capture Format: Direct probe capture (cp) Seci ID No 14 immobilized on the probe membrane; Detection probe: via 3 nucleotides (N3), 6 nucleotides (N6), S, Or the SS spacer is linked to the 5'-end of the detection probe (dp) Seci ID No 13, Seq ID No 15 and Seq ID No 16. Each l〇12 copy. Detection format: anti-biotin antibody-dye conjugate; target DNA: 214 bp double strand DNA fragment, 1011 - 101G replica. -21 - 1286159 V. INSTRUCTIONS (20) A copy of the result target: 1011 5x10 丨0 10丨0 Detection probe signal jjhuang dpl3-B+dpl5-B+dpl6-B 1.5 1.0 0.0 dpl3-N3-B+ dpl5-N3-B+dpl6-N3-B 3.0 2.0 0.5 dpl3-N6-B+dpl5-N6-B+dpl6-N6-B 4.5 3.0 1.0 dpl3-S-B+dpl5-S-B+dpl6-SB 3.0 2.0 <0.5 dpl3-SS-B+dpl5-SS-B+dpl6-SS-B 3.5 3.0 0.5 dpl3 No identification +dpl5-N6-B+dpl6-N6-B 2.5 1.5 0.5 These results show: Use N3, N6 The S, or SS spacer can improve the detection sensitivity of the double-stranded target nucleic acid by more than 5 times. The N6 and SS spacers have higher detection sensitivity than the N3 and S spacers, indicating that the spacer length is important for the improvement of detection sensitivity. The N6 spacer has higher detection sensitivity than the SS spacer. Despite the fact that these spacers are of equal length, it is important that the spacer physicochemical properties are improved for detection sensitivity. Use only two detection probes to connect biotin to the 5'-end with N6 spacer (#13 no marker + (1?15-~^ + (^16-~^)) (where biotin is located in capture probe The detection sensitivity of the internal orientation of the needle-capture complex is greater than the direct connection of biotin to the 5'-end using three detection probes (dp 13- -22- 1286159. V. Description of invention (21) B + dpl5-B + dpl6-B) (where one of the detection probes (dpl3-B) biotin is located in the external orientation of the complex captured by the capture probe). Example 1 and Example 2 Conclusion The sensitivity of target nucleic acid detection can be increased by using a spacer to detect the ligand to the detection probe. Longer spacers are better than short spacers. Intervals of the same length but different physicochemical properties Sensitivity has different effects. Especially the non-protein components in the spacer are composed of only nucleotides, preferably those with non-nucleotide components in the spacer. Possible explanations for these results: 1. Nucleotide spacer Improved detection sensitivity may be through the hybridization of the detection probe to the target nucleic acid. When hybridizing to a target nucleic acid, the nucleotides of these spacers are not expected to base pair with the nucleotide of the target nucleic acid. The nucleobases of these nucleotides can be hybridized to the target pair (formed on the detection probe) The nucleic acid forms a stacking interaction. These stacking interactions enhance the stability of the hybrid (hyb I* id ) formed between the target nucleic acid and the detection probe, thereby enhancing the detection sensitivity of the target nucleic acid. The nucleoside spacer is harder than the S and SS spacers. The ribose ring of the nucleotide spacer is expected to provide more robustness than the polyethylene glycol group of the S and SS spacers. Larger hardness may increase. Detecting the availability of a ligand using a linked nucleotide spacer that interacts with a portion that detects the binding of the ligand. 3. The polarity between the nucleotide and the non-nucleotide spacer may cause detection - 23- 1286159 V. INSTRUCTIONS (22) The sensitivity of the measurement is different. Example 3 Experimental composition Capture format: Direct probe capture (cp) fixed to the probe (Se) ID No 10; Detection probe: directly or via N6 , SS, (ds) 6, (Sc3) 6 or SNJNJ interval Connect biotin to the 5'-end of the detection probe (dp) seq id No 13. Each copy of 1 012. Detection format: anti-biotin antibody-dye conjugate; helper probes: SEQ I]) No 5 and SEQ ID No 6 are adjacent to SEQ ID No 10; SEQ ID No 1 and SEq π No 2 are adjacent to SEQ ID No 13, 1012; target DNA: 872 bp double-stranded DNA fragment: 410^-5 ^101° Replica of the target result: 2xl〇" 5xl〇10 Detection probe signal strength dp-B <1.0 0.0 dp-N6-B 2.0 0.5 dp-SS-B 1.0 0.0 dp-(dS)6- B 1.0 0.0 dp- (SC3)6-B 1.0 0.0 dp-SN3SN3S-B 1.5 0.0 -24- 1286159 V. INSTRUCTIONS (23) These results show: despite the SS '(dS)6 and (sc3)6 spacers The structural differences and properties are identical in length and have similar effects in enhancing the detection sensitivity of the target nucleic acid. The I spacer has the same length as the SS, (dS)6 & (SC3)6 spacer, whereas the N6 spacer has the greatest effect on improving the detection sensitivity of the target nucleic acid. The use of N6 spacers has a greater target nucleic acid detection sensitivity than the sn3sn3s spacer (the longest test spacer). This situation may be explained by the fact that the S monomer reduces or eliminates the stacking interaction between the nucleobase and base pairs of the spacer N3 component (formed between the detection probe and the target nucleic acid). This data supports the conclusion that the stacking interaction of the unpaired nucleobases of the spacer with the dimers formed between the target nucleic acid and the detection probe is important in enhancing the detection sensitivity of the target nucleic acid. Example 4 Spacers having different physicochemical properties and lengths were evaluated by a stick test and a dot blot analysis. In the absence of detection probe hybridization to the target nucleic acid, the dot blot assay enables efficient interaction of the avidin antibody with the avidin-linked detection probe to be analyzed. 5 X 1 08 - 5 X 1 011 Replica The anti-biotin detection probe was attached at different intervals, spotted at different positions of the normal-priced nylon membrane, and UV-crosslinked to the membrane. The membrane is then incubated with an alkaline phosphatase (an oxon tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BC IP) chromogenic substrate) And cultivating NBT/BCIP colorimetric receptors, and then observing whether the film saw any color formed in the capture zone. -25- 1286159 V. INSTRUCTIONS (24) Experimental setup of the rod test capture format: direct probe capture (cp) Seq ID No 14 fixed in the probe; detection of the I probe · directly or via nucleotides Or a non-nucleotide spacer to link biotin to the 5'-end of the detection probe (dp) Seq ID No 13. Each 1012 replica. Detection format: anti-biotin antibody-dye conjugate; helper probe: SEQ ID No 2 and SEQ ID No 3 adjacent to SEQ ID No 14, 1〇12 copy; target DNA: 416 bp double strand DNA fragment, 5xl01G- 5xl09 replica. Result Replica of the target: 2χ10η 5χ101ϋ Detection probe Signal strength
dp - Β dp -N6-B d p - S S - Β dp - (dS ) 6-B dp-(SC3)6-B dp-SN3SN3S-BDp - Β dp -N6-B d p - S S - Β dp - (dS ) 6-B dp-(SC3)6-B dp-SN3SN3S-B
點墨分析之實驗構成 偵測探針：直接或經由核苷酸或非核苷酸間隔子連接生 -26- 1286159 五、發明說明（25) 物素到偵測探針（dp) SeQ ID N〇 13之5’-端。 偵測格式：抗-生物素抗體連接驗性憐酸酶，經由 NBT / BC I P產色性受質偵測。 結果 間隔子 Μ j\\\ N3 n4 n5 n6 (dS)6 SN3SN3S ssss sss ss s 偵測限制 5.0 x Ell 5.0 x El 0 5.0 x E10 5.0 x El0 2.5 x E10 2.5 x El 0 2.5 x E9 2·5 x E9 5·0 x E9 2.5 x E10 5.0 x Ell 檢榛測試之結果顯示匕 使用含有3，4或5核苷酸間隔子之偵測探針’於目標 核酸的偵測敏感度上無顯著不同。 含有6核苷酸間隔子的偵測敏感度勉強較佳於3 - 5核苷 酸間隔子。 -27- 1286159 五、發明說明（26) 含有僅由核苷酸構成之間隔子的偵測敏感度較佳於含有 非核苷酸之間隔子。例如n3間隔子較佳於最長的sn3sn3s 間隔子，相當於1 5核苷酸長度。 (d S ) 6間隔子稍稍好於最長的S S間隔子。 y墨分析之結果顯示： 含有最長間隔子（SSSS及SN3SN3S)的偵測敏感度最高。 使用相同長度、不同物化性質之間隔子（N6，（(^)6及SS) 的偵測敏感度則類似。 眚施例3及4之結論 dS成分具有核苷酸的類似結構。二者皆有核糖殘基，預 測可提供堅硬度。然而，dS成分不含核苷酸擁有之核鹼基 。（dS ) 6間隔子相較於N6間隔子在目標核酸偵測之敏感度 的不同影響，推測說明使用核苷酸間隔子在偵測敏感度之 改良有較大影響，主要係因不與目標核酸鹼基配對之核鹼 基的存在。 間隔子的組成物顯得比其長度更重要（比較實施例4之 檢棒測試中#-1-：85’及〇1?-31313-35’的結果）。 使用（dS ) 6間隔子的偵測敏感度大於SS間隔子。這些間 隔子長度相同。就此推測間隔子的物化性質’例如堅硬度 或極性可能在間隔子改良偵測敏感度上也有影響。 實施例4之點墨分析顯示間隔子長度對於生物素對抗生 物素抗體的可獲性很重要。以抗生物素抗體辨認生物素的 敏感度皆類似，不論生物素是否經由N6 ’（dS)6或SS間隔 -28- 1286159 五、發明說明（27) 子連接到被固定之探針。然而這些間隔子於實施例4之檢 棒測試中對偵測敏感度的影響不同。就此推測在偵測探針 對目標核酸之雜交作用中，間隔子之組成物比抗生物素抗 體對生物素之辨認作用中更重要。以抗生物素抗體（或其 他偵測配位體結合部分）辨認生物素（或其他偵測配位體） 時，間隔子長度顯得更重要於間隔子之組成物。 實施例5 眚驗構成 捕獲格式：直接探針捕獲（cp) Seq ID No 14連接固定 在檢棒之BSA。捕獲探針直接或經由6核苷酸間隔子連接 BSA ； 偵測探針：連接螢光素之偵測探針（d p ) S e q ID Ν ο 1 3 。1 0 12複本。 偵測格式：抗-生物素抗體-染料結合物； 目標DNA : 73 nt或76 nt之單股DNA片段，1011複本 結果 目標之複本： 10丨1 捕獲探針 訊號強度 cp-BSA-檢棒 4.0 cp-N6-BSA-檢棒 5.0 -29- 1286159The experiment of spot ink analysis constitutes a detection probe: directly or via a nucleotide or non-nucleotide spacer. -26- 1286159 V. Description of the invention (25) Element to detection probe (dp) SeQ ID N〇 The 5'-end of 13 Detection format: anti-biotin antibody linked to the detection of pity acid, detected by NBT / BC I P colorimetric receptor. Result interval Μ j\\\ N3 n4 n5 n6 (dS)6 SN3SN3S ssss sss ss s Detection limit 5.0 x Ell 5.0 x El 0 5.0 x E10 5.0 x El0 2.5 x E10 2.5 x El 0 2.5 x E9 2·5 x E9 5·0 x E9 2.5 x E10 5.0 x Ell The results of the test showed no significant difference in the detection sensitivity of the target nucleic acid using a detection probe containing a 3, 4 or 5 nucleotide spacer. . The detection sensitivity of the 6-nucleotide spacer is barely preferred to the 3 - 5 nucleotide spacer. -27- 1286159 V. DESCRIPTION OF THE INVENTION (26) The detection sensitivity of a spacer containing only nucleotides is preferably a spacer containing a non-nucleotide. For example, the n3 spacer is preferably the longest sn3sn3s spacer, corresponding to a length of fifteen nucleotides. The (d S ) 6 spacer is slightly better than the longest S S spacer. The results of the y ink analysis show that the longest interval (SSSS and SN3SN3S) has the highest detection sensitivity. The detection sensitivity of the spacers of the same length and different physicochemical properties (N6, ((^)6 and SS) is similar.) The conclusions of Examples 3 and 4 The dS component has a similar structure of nucleotides. There are ribose residues that are predicted to provide firmness. However, the dS component does not contain nucleotide-owned nucleobases. The (dS) 6 spacers have different effects on the sensitivity of target nucleic acid detection than the N6 spacers. It is speculated that the use of nucleotide spacers has a greater impact on the improvement of detection sensitivity, mainly due to the presence of nucleobases that are not paired with the target nucleic acid base. The composition of the spacer appears to be more important than its length (compare The results of #-1-:85' and 〇1?-31313-35' in the stick test of Example 4) The detection sensitivity of the (dS) 6 spacer is greater than that of the SS spacer. These spacers are the same length. In this regard, it is speculated that the physicochemical properties of the spacers such as hardness or polarity may also have an effect on the improved sensitivity of the spacer detection. The dot blot analysis of Example 4 shows that the spacer length is very compatible with biotin against biotin antibodies. Important. Identify with anti-biotin antibodies The sensitivity of biotin is similar, regardless of whether biotin is attached to the immobilized probe via N6 '(dS)6 or SS spacer -28-1286159 5. Invention note (27). However, these spacers are used in Example 4. The effect of detection on the detection sensitivity is different. It is speculated that in the hybridization of the detection probe to the target nucleic acid, the composition of the spacer is more important than the recognition of biotin by the anti-biotin antibody. When biotin antibodies (or other detecting ligand binding moieties) recognize biotin (or other detecting ligands), the spacer length appears to be more important to the spacer composition. Example 5 The assay constitutes the capture format: Direct probe capture (cp) Seq ID No 14 is ligated to the BSA of the probe. The capture probe is ligated directly or via a 6 nucleotide spacer to the BSA; detection probe: a detection probe for luciferin attachment (dp) S eq ID Ν ο 1 3 .1 0 12 copy. Detection format: anti-biotin antibody-dye conjugate; target DNA: 73 nt or 76 nt single-strand DNA fragment, 1011 replica result copy of target: 10丨1 capture probe signal strong cp-BSA- subject bar 4.0 cp-N6-BSA- subject -29-1286159 5.0 bar
五、發明說明（28) 實施例6 實驗構成 捕獲格式：直接探針捕獲Up) Seq ID No 14在5，_端 連接固定在檢棒之BSA。捕獲探針經由n6或SN3SN3S間隔 子連接BSA ; 偵測探針：連接生物素之偵測探針（d p ) s e q I D N 〇 7， 8，9，10，11，12，13，15，16 及 17。每一各 ι〇12 複本 偵測格式：抗-生物素抗體-染料結合物； 目標DNA : 872 bp之雙股DNA片段，1 〇 11 _ 2 . 5x 1 09複本 〇 結果 目標之複本： 1011 2.5xl〇10 l〇10 5xl09 2.5xl09 捕獲探針 訊號強度 cp-SN3SN3S-BSA-檢棒 4.0 3.5 2.5 1.0 0.0 cp-N6-BSA_ 檢棒 4.5 4.0 3.0 1.5 0.0 實施例7 實驗構成 捕獲格式：直接探針捕獲（cp) Seq ID No 15連接固定 在檢棒之BSA。捕獲探針經由n6間隔子連接bsA ; 幫手探針：SEQ ID No 3 及 SEQ ID No 4 鄰近 SEQ ID -30- 1286159 五、發明說明（29) No 15 ； 偵測探針··此實施例中偵測探針含有吊鉤探針及一般探 針。吊鉤探針具有序列對應於S e q I D No 1 7 (能雜交到目 標核酸）以及序列互補於一般探針之序列。一般探針經由 …或SN3SN6間隔子連接紡織染料。吊鉤探針有i〇i2複本。 目標：872 bp雙股DNA片段，1011及101°複本。 結果 目標之複本： 1011 1010 捕獲探針 訊號強度 一般探針-SN3SN6-染料 2.0 0.0 一般探針-N6-染料 2.0 0.5 實施例5，6及7之結論 使用含有固定在檢棒之蛋白質以及連接捕獲探針至固定 蛋白質（實施例5及6 )之非蛋白質之間隔子，或使用非蛋 白質間隔子連接標識至偵測探針（實施例7 )能改良目標核 酸偵測之敏感度。 當間隔子非蛋白質成分完全由核苷酸構成時，目標核酸 偵測之敏感度最高。 檢棒之單股及雙股目標核酸之偵測敏感度，根據本發明 之非配對核苷酸之使用發現具有顯著改良。雙股環狀目標 核酸之偵測敏感度也能根據本發明之非配對核苷酸之使用 -31 - 1286159 五、發明說明（3〇) 而增加。 已知單股目標核酸可藉由分子內鹼基配對交互作用形成 二級結構。如此二級結構能抑制捕獲探針及偵測探針對目 標核酸之結合。因此，偵測探針及捕獲探針結合處之目標 核酸區域，常選爲預期實質上無二級結構之區域。 雙股目標核酸之偵測敏感度的改良，可根據本發明之非 配對核苷酸之使用達成，意指單股目標核酸之偵測敏感度 使用捕獲探針及/或偵測探針（能辨認含有二級結構之目標 核酸區域）亦獲改良。此項優點爲捕獲探針及/或偵測探針 之選擇不需根據目標核酸形成二級結構之預測，如此簡化 捕獲及偵測探針之選擇。 檢棒偵測之傳統方法在環狀雙股目標核酸之偵測上非常 不足。因此，此等目標核酸通常在偵測之前處以酵素將雙 股目標直線化。環狀雙股目標核酸之偵測改良可根據本發 明之非配對核苷酸之使用而達成，意指不需要目標核酸的 直線化作用而簡化偵測方法。 元件符號對照片 10 檢棒 12 目標核酸 14 樣品溶液 16 硝基纖維片 18 接觸端 20 捕獲探針 -32- 1286159 五、 發明說明（ 31 ) 22 捕獲區 24 抗-生 物素抗體-染料結合物 26 結合區 28 偵測探 針 -33-V. INSTRUCTIONS (28) Example 6 Experimental Composition Capture Format: Direct Probe Capture Up) Seq ID No 14 is connected to the BSA fixed to the detector at the 5, _ end. The capture probe is ligated to the BSA via the n6 or SN3SN3S spacer; the detection probe: the biotin detection probe (dp) seq IDN 〇7, 8,9,10,11,12,13,15,16 and 17 . Each ι〇12 duplicate detection format: anti-biotin antibody-dye conjugate; target DNA: double-stranded DNA fragment of 872 bp, 1 〇11 _ 2 . 5x 1 09 duplicate 〇 copy of the result target: 1011 2.5 Xl〇10 l〇10 5xl09 2.5xl09 Capture probe signal strength cp-SN3SN3S-BSA-Checker 4.0 3.5 2.5 1.0 0.0 cp-N6-BSA_ Checker 4.5 4.0 3.0 1.5 0.0 Example 7 Experimental composition Capture format: Direct probe Capture (cp) Seq ID No 15 is connected to the BSA fixed to the detector. The capture probe is ligated to bsA via an n6 spacer; the helper probe: SEQ ID No 3 and SEQ ID No 4 adjacent to SEQ ID -30- 1286159 5. Inventive Note (29) No 15 ; Detection Probe·· In this embodiment The detection probe contains a hook probe and a general probe. The hook probe has a sequence corresponding to S e q I D No 1 7 (which can hybridize to a target nucleic acid) and a sequence complementary to a general probe. Typically the probe is attached to the textile dye via a ... or SN3SN6 spacer. The hook probe has a copy of i〇i2. Target: 872 bp double strand DNA fragment, 1011 and 101° replica. Replica of the target: 1011 1010 Capture probe signal intensity General probe - SN3SN6 - Dye 2.0 0.0 General probe - N6 - Dye 2.0 0.5 Examples 5, 6 and 7 Conclusions Using proteins containing immobilized probes and connection capture The non-protein spacer of the probe to the immobilized protein (Examples 5 and 6) or the non-protein spacer linkage to the detection probe (Example 7) improves the sensitivity of the target nucleic acid detection. When the spacer non-protein component is composed entirely of nucleotides, the target nucleic acid is most sensitive to detection. The detection sensitivity of the single and double-stranded target nucleic acids of the test strips was found to be significantly improved by the use of the unpaired nucleotides of the present invention. The detection sensitivity of the double-stranded circular target nucleic acid can also be increased according to the use of the unpaired nucleotide of the present invention - 31 - 1286159 V. Description of the invention (3〇). Single-stranded target nucleic acids are known to form secondary structures by intramolecular base pairing interactions. Such a secondary structure inhibits binding of the capture probe and the detection probe to the target nucleic acid. Therefore, the target nucleic acid region at the junction of the detection probe and the capture probe is often selected as the region where the secondary structure is expected to be substantially absent. The improvement of the detection sensitivity of the double-stranded target nucleic acid can be achieved according to the use of the unpaired nucleotide of the present invention, meaning that the detection sensitivity of the single-stranded target nucleic acid uses a capture probe and/or a detection probe (can The recognition of the target nucleic acid region containing the secondary structure has also been improved. The advantage of this is that the selection of the capture probe and/or the detection probe does not require the prediction of a secondary structure based on the target nucleic acid, thus simplifying the selection of the capture and detection probes. The traditional method of detecting rods is very inadequate in detecting circular double-stranded target nucleic acids. Therefore, these target nucleic acids are usually linearized with enzymes prior to detection. The detection improvement of the circular double-stranded target nucleic acid can be achieved according to the use of the unpaired nucleotide of the present invention, meaning that the linearization of the target nucleic acid is not required to simplify the detection method. Component symbol pair photo 10 check rod 12 target nucleic acid 14 sample solution 16 nitro fiber sheet 18 contact end 20 capture probe -32- 1286159 V. Description of invention (31) 22 Capture zone 24 Anti-biotin antibody-dye conjugate 26 Binding zone 28 detection probe-33-