1262947 九、發明說明： 【發明所屬之技術領域】 本發明係有關一種病毒之檢驗技術，詳言之，係有關一 種以即時聚合酶連鎖反應定量病毒之檢驗技術。 【先前技術】 台灣地區是B型肝炎病毒的高盛行區，於4〇歲以上的台灣 健康人口中’ B型肝炎病毒的感染率將近有15至20%，目前 約有三百萬人為B型肝炎帶原者，佔國人十大死亡原因首位 之肝細胞癌及第六位之慢性肝炎及肝硬化，皆與B型肝炎病 毋感染相關。B型肝炎病毒會在宿主的肝細胞内複製，並釋 出表面抗原（HBsAg)、e抗原（HBeAg)以及含有DNA的完整 病毒顆粒到血液中。 現今臨床上B型肝炎帶原者之診斷是藉鑑定血清HBsAg 是否呈陽性反應而決定；如為慢性持續的感染，通常血清 DNA或HBeAg亦呈陽性反應；如在緩解的階段，則血清DNA 或HBeAg則呈陰性反應，此種血清學的變化會同時伴隨著 肝組織以及肝功能的改善；再者，可藉由偵測血清中是否 有B型肝炎病毒的DNA，以評估病毒是否處於活躍的複製 期，然而於肝炎的治療過程令，因為肝炎患者並無明顯的 臨床症狀，亦必須依賴人體血液中肝炎病毒濃度之偵測， 做為臨床診斷的依據。 綜上所述’偵測血中病毒濃度於診型肝炎或追蹤抗病 毒藥物之效果而言，乃十分重要。 目前’B型肝炎病毒量的測定方法，包括各研究單位自創 96645.doc 1262947 的競爭性核酸聚合酶連鎖反應及即時偵測聚合酶連鎖反 應’與商品化的Amplicormonitor(R〇cheTM Diagnostics，巴 賽爾’瑞士）、Branched-chain DNA(Bayer™ Diagnostics， 艾莫利威爾，美國）及NASBA (〇rganon TeknikaTM，博克斯 特爾，荷蘭）等方法。然而上述商品具有下述缺點··（丨）操作 耗時，步驟繁瑣；（2)檢測之線性範圍不佳；（3)無樣品内部 品管。 職是之故，發展一操作容易、靈敏度高、可靠性高之b 型肝炎檢測技術乃為業界所需。 【發明内容】 發明概述 本發明之一目的在於提供-種用於定量B型肝炎病毒之 引子，其可放大B型肝炎病毒DNA，該引子係選自由下列引 子及其變異體所組成之群:具有如序列辨識編號嘀示序列 之一上游引子、具有如序列辨識編號3所示序列之—上游引 子、具有如序列辨識編號4所示序列之-上游引子、具有如 序列辨識編號2所示序列之一 /、 心„ , J之下游引子、具有如序列辨識編 唬5所不序列之一下游引早 子及具有如序列辨識編號ό所示序 列之一下游引子。 本發明之另一目的在於裎极 隹於k仏一種用於定量β型肝炎病毒 之套組，其包含如上述之引子。 本發明之又一目的扃於植 在於如I、一種定量Β型肝炎病毒方 法，其係使用上述引子推广 子進仃即時聚合酶連鎖反應。 發明詳細說明 96645.doc 1262947 本發明係關於一種用於定量B型肝炎病毒之引子，其可應 用於各式即時聚合酶連鎖反應，並藉以定量b型肝炎病毒。 根據本發明之引子可放大B型肝炎病毒圓八，該引子丙:選 自由下列引子及其變異體所組成之群：具有如序列辨識編 號1所：序列之-上游引子、具有如序列辨識編號3所示序 列之一上游引子、具有如序列辨識編號4所示序列之一上游 引子、具有如序列辨識編號2所示序列之一下游引子、具有 如序列辨識編號5所示序列之一下游引子及具有如序列辨 識編號6所示序列之一下游引子。較佳地，根據本發明之引 子係形成下列引子對： (a) 具有如序列辨識編號1所示序列之一上游引子及具 有如序列辨識編號2所示序列之一下游引子· (b) 具有如序列辨識編號3所示序列之一上游引子及具 有如序列辨識編號5所示序列之一下游引子；及 (c) 具有如序列辨識編號4所示序列之一上游引子及具 有如序列辨識編號6所示序列之一下游引子。 本文中所言之「聚合酶連鎖反應」包含四個步驟··（1)使 1模板進行變性’以形成兩單股；⑺使兩引子分別與步驟 (1)之兩股進行黏附（annealing) ; (3)以DNA聚合酶延伸該等 引子，及（4)取得兩雙股之DNA。重複上述之諸等步驟，而 :特定之DNA片段即可獲得擴增。「即時聚合酶連鎖反應」 二為利用螢光偵測。「聚合酶連鎖反應」之發生以進行定 人並可彳欠而推知參與反應之模板量。目前發展之即時聚 口酶連鎖反應是在一個封閉式的反應管中，除了聚合酶連 96645.doc 1262947 鎖反應所而的引子與驗基外，—併將螢光訊號加人反應管 中再配&儀為逐週期摘測，便可達到同時擴增且同 時傾測訊號之功能。故即時聚合酶連鎖反應具有擴增完成 可直接定量及不需後處理之優點，不僅省時省力，更可避 免後處理所&成之，亏染。同時亦因逐週期紀錄的η·榮光 訊號，同時描繪出基因複製時之完整圖形，故具有較好之 靈敏度與再現性。本發明之試劑濃度線性範圍可達⑻至1〇8 複本數，為現行方法中最大者。 變異體」乙詞係指可取代本發明引子 而仍可放大同一特定片段序列之引子 中 本文中所言之「 之募核芽酸分子， 溫度越低；反言之，如引子與欲放大之模板間之序列變異 因聚合酶連鎖反應本身之特性，引子與欲放大之模板間之 序列即便存在變異性，仍可藉調節聚合_鎖反應中黏附 步驟之反應溫度而合成特定之DNA片段。舉例言之，如引 子與欲放大之模板間之序列變異性越大，黏附步驟之反應 性越小，則可提高黏附步驟之反應溫度。故於該特定領域 中八般知識之人士根據本發明之揭示，即可根據欲增幅 放大之DNA片段設計不同之引子，任何針對本發明引子之 鹼基置換、加入或縮減，如其仍可與本發明引子放大特定 片段之引子皆為本發明所欲保護之範圍。 根據本發明之引子可應用於纟式即時聚合酶連鎖反應 中，舉例言之，可使用於DNA結合螢光基（DNA-binding flU〇r〇Ph〇reS)、鄰近線性募探針（adjacent linear 〇lig〇pr〇bes)、 5核酸酶寡探針nuclease 〇lig〇pr〇bes)、髮夾式募探針 96645.doc 1262947 (hairpin oligoprobes)及自我螢光擴增子（self-fluorescing amplicons)等系統。 本發明另提供一定量B型肝炎病毒之套組，其包含上述之 引子或引子對。 於本發明之一具體貫施例中’係使用TaqMan®即時聚合 酶連鎖反應系統。該系統係藉著一目標探針以定量聚合酶 連鎖反應產物。該目標探針於5,及3,端各分別具有作為報導 劑及泮滅劑之兩螢光基。該螢光基為報導蛋白質之螢光部 分’通常使用之螢光基為FAM。如目標探針未與模板雜合 時，其位於3’端之淬滅劑螢光基（通常為一長波長顏色，如 紅色）藉著螢光共振能量轉移（Fluorescence Res()nanee Energy Transfer，FRET)而減小5’端之報導劑螢光基（通常為 一短波長顏色，如綠色）之螢光。如目標探針於模板變性而 黏附至單股模板上時’叫聚合酶可一面依模板之序列加上 核苦酸’ -邊自5,端移除目標探針，因而使3ι端之泮滅劑榮 光基與5’端之報導劑分離，而使報導劑可發散其螢光能量， 如聚合酶連鎖反應發生之次數愈多，則有愈多之報導劑螢 光發散，故藉著量測報導劑螢光之量，即可定量聚合酶連 鎖反應之產物。故根據本發明之套組，另包含_目標探針， 該目標探針係可與根據本發明之引子或引子對所^大之B 型肝炎病毒亀片段雜合，且其5，及3，端分別具有_第一報 導劑及一第一泮滅劑。於本發明中所使用之第-報導劑盥 弟-泮滅劑可為習用之螢光基，如使_ 劑。較佳地，當使用⑷引子對時，該目標探針具有如序^ 96645.doc 1262947 辨識編號7或其互補股所示之序列。 =認量測所得之訊號係為反應真實之實驗條件或是操 :乍：差，根據本發明之套組另包含一内部控制機制，其包 二内部控制質體，其具有一插入片段，該插入片段包含 ::與B型肝炎DNA或與人類基因體序列皆無相似性片段 ，由根據本發明之引子或引子對所放大之_肝炎病 毋DNA片段；及一内部控制探針，可與該内部控制質體之 =段雜合，其5,及3,端分別具有一第二報導劑及一第二 =;且該第二報導劑與該第-報導劑不同。於操作時 控制質體與㈣控制探針之即時聚合酶連鎖反應與 樣°°及目標探針同時操作，以便於觀察。於本發明中所使 用之苐一報導劑與第二淬滅劑可為習用之螢光基，如使用 VK：作為弟—報導劑。較佳地，該插人片段具有如序列辨識 編號12或其互補股所示之序列，且該内部控制探針具有如 序列辨識編號10或其互補股所示之序列。 :本發明之另一具體實施例中，係使用雜合探針即時聚 合酶連鎖反應系統。該系統係藉著一定錯探針及—感應探 針以定量聚合酶連鎖反應產物。該感應探針U端具有一施 體螯光、，該定錫探針之5,端具有—受體螢光。施體榮光可由 適田之波長所激♦，並可將能量轉移給受體螢光，而經激 發之受體勞光則可發散出—較長之波長。如定錯探針及咸 應探針未與模板雜合時，於適當波長激發下，可發散出施 體榮先’但由於其未鄰近受體榮光，而無法將能量轉移， 故只可觀察得施體螢光之量。如定錯探針與感應探針於模 96645.doc 10 1262947 板變性而黏附至單股模板上時，施體螢光即可將能量移轉 至受體螢光，故可觀察到受體螢光之量，如聚合酶連鎖反 應發生之次數愈多，製造愈多產物做為模板，則有愈多之 施體螢光轉移至受體螢光，而可觀察到受體螢光發散，故 藉著置測受體螢光之量，即可定量聚合酶連鎖反應之產 物。故根據本發明之套組，其另包含一感應探針及一定錯 探針，該感應探針之3,端具有一第一施體螢光，該定錨探針 之5’端具有一第一受體螢光，且該感應探針與定錨探針可分 別與根據本發明之引子或引子對所放大之B型肝炎病毒 DNA片段雜合，且當該定錨探針與感應探針同時雜合至根 據本發明之引子或引子對所放大之B型肝炎病片段 時，可使第一施體螢光之能量移轉至第一受體螢光。於本 發明中所使用之第一受體螢光與第一施體螢光可為習用之 螢光基，如使用FL作為第一施體螢光，使用[C Red^仂 為第-受體螢光。較佳地，當使用⑻或⑷引子對時， 忒感應探針具有如序列辨識編號8或其互補股所示之 列，且該定錨探針具有如或序列辨識編號9或其互補股所示 頰似地，本具體實施例亦包含 g σ丨炫咧機制，苴勹a α之一内部控制質體及-内部控制定錨探針，龙5•戚= 一第二受體螢光並可與内部控制質體之插人有 當該内部定錨探針與感應探針 又…’且 時’可使第-施體螢光之能量移二 作時將内部控制質體與内部定㈣及感應探::即= 96645.doc 1262947 合酶連鎖反庫盥媒σ 樣叩及疋錨探針及感 便於觀察。較佳祕，姑h 木对I』時知作，以 ^ # 11 i? JL ^部控制定錨探針具有如序列辨識 、，扁油或其互補股所示之序列。 較佳地，根據本發明之套組，其另 作桿準曲絲甘+ &準貝體用以 ，卓曲線’丨中該標準質體具有一外加片段 奴包含由根據本發明^ ^ ^ ^ ^ ^ ^ ^ ^ Λ 毒dna月卜… 對所放大之_肝炎病 :標準質體之其他部分構築之” 明所屬技術領域t具一般 、’、’、本《 體實施例中該外加& 於—較佳具 所示之序列。 具有如序列辨識編號13或其互補股 ^發明之套組與所有全《型肝炎的核酸序列掏取 證實根據本發明之套組可以辨識現有已知基因型， :免發生因為病毒基因型或類種差異而造成之偽陰 檢測率達到最高。 便 本發明再關於-種用於定量B型肝炎病毒之方法，盆係使 隸據本發明之引子或套組進行即«合酶連鎖反應。 誶言之’當使用TaqMan®即時聚合酶連鎖反應系統時， 根據本發明之方法包含下列步驟： ⑴以不同濃度之標準質體與一目標探針進行雜合反 應，該標準質體具有-外加片段，該外加片段包含由 根據本發明之引子或引子對所放大之B型肝炎病毒 DNA片段，且該目標探針係可與該根據本發明之引子 或引子對所放大之B型肝炎病毒DNA片段雜合，該目 標探針之5，及31端分別具有—第一報導劑〗一第一泮 96645.doc 1262947 滅劑； (II) 里測步驟⑴中以不同濃度之標準質體反應中第一報 導悧之1，以得到一標準質體濃度對第一報導劑量之 標準曲線； (III) 彳乂得衍生自樣品之B型肝炎病毒之;較佳地，樣 品係源自血液，如血清或血漿； (·)、丨子或引子對、目標探針、及步驟（出）中之DM A進 行即時聚合酶連鎖反應； ()里成I步驟（lv)中第一報導劑之強度，並與步驟（ϋ)中所 传之標準曲線比較，以定量樣品中DNA之濃度。 較佳地，根據本發明之方法包含一内部控制步驟，係加 入一内部控制質體與—内部控制探針，並與b型肝炎病毒 臟及目標探針進行步驟（iv)及⑺，並量測第二報導劑之 量Ή貞測得陽性訊號’則代表反應條件成功。 詳言之，當使㈣合探針即時聚合酶連鎖反應系統時， 根據本發明之方法包含下列步驟： ⑴以不同濃度之標準質體與一感應探針及一定錯探針 進行雜合反應；該標準質體具有-外加片段，該外加 片段包含由根據本發明之引子或引子對所放大之β 型肝炎病毒DNA片段；該感應探針之3,端具有一第一 施體螢光，該定錯探針之5，端具有一第一受體營光， 該感應探針與定錯探針可分別與根據本發明之^子 或引子對所放大之3型肝炎病毒而八片段雜合，且合 為疋ί“木針與感應探針同時雜合至根據纟發明之引 96645.doc 1262947 子或引子對所放大之B型肝炎病.DNA片段時，可使 第轭體螢光之能量移轉至第一受體螢光； (11)量測步驟⑴中以不同濃度之標準質體反應中第一受 體螢光之量，以得到一標準質體濃度對受體螢光量之 標準曲線； (Hi)獲得衍生自樣品之B型肝炎病毒之DNA;較佳地，樣 品係源自血液，如血清或血漿； ㈣以根據本發明之引子或引子對、感應探針、定錯探 及乂驟⑴丨）中之DNA進行即時聚合酶連鎖反應； 00量測步驟（iv)中受體螢光之強度，並與步驟⑼中所得 之標準曲線比較，以定量樣品中DNA之濃度。 較佳地’根據本發明之方法包含一内部控制步驟，係加 入-内部控制質體及—内部控制定錨探針，並與B型肝炎 撕二及^錨探針進行步驟㈣及⑺，並量測第二受體營光 之量，如制得陽性域，則代表反應條件成功。 於本發明中，樣品中病毒黯之萃取、即時聚合酵素連 鎖反應以及訊號之分析，係藉由熟習該項技術者所熟知之 方法而達成，較佳的樣品中，病毒DNA之萃取、即時聚合 酵素連鎖反應以及訊號之分析說明於以下的實例中。 兹以下列實例予以詳細說明本發明，唯並不意味本發明 僅侷限於此等實例所揭示之内容。 【實施方式】 實例一 ·· B型肝炎DNA之製備 血清檢體保存步驟： 96645.doc -14· 1262947 -使用不含抗旋劑的抽血管採集病人的血液，約丨〇至丨5分 鐘待血液凝固； -以3000 rpm離心10分鐘（室溫兩小時内完成） -吸取上層血清 -分裝50 μί於微量離心管中1262947 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a virus detection technique, and more particularly to an assay technique for quantifying a virus by an instant polymerase chain reaction. [Prior Art] Taiwan is a high-risk area of hepatitis B virus. In the healthy population of Taiwan over 4 years old, the infection rate of hepatitis B virus is nearly 15 to 20%. At present, about 3 million people are type B. Hepatitis with the original, the first cause of death in the country's top ten causes of hepatocellular carcinoma and the sixth chronic hepatitis and cirrhosis, are associated with hepatitis B infection. Hepatitis B virus replicates in the liver cells of the host and releases surface antigen (HBsAg), e antigen (HBeAg), and intact viral particles containing DNA into the blood. In today's clinical diagnosis, the diagnosis of hepatitis B is based on whether the serum HBsAg is positive or not; in the case of chronic persistent infection, serum DNA or HBeAg is also positive; if at the stage of remission, serum DNA or HBeAg is negative, and this serological change is accompanied by improvement of liver tissue and liver function. Furthermore, it can be evaluated whether the virus is active by detecting whether there is DNA of hepatitis B virus in the serum. The replication period, however, in the treatment of hepatitis, because hepatitis patients have no obvious clinical symptoms, must also rely on the detection of hepatitis virus concentration in human blood, as a basis for clinical diagnosis. In summary, it is important to detect the effect of virus concentration in blood on the diagnosis of hepatitis or to track anti-viral drugs. The current method for determining the amount of hepatitis B virus, including the competitive nucleic acid polymerase chain reaction and the instant detection of polymerase chain reaction of each research unit, 96645.doc 1262947, and the commercial Amplicormonitor (R〇cheTM Diagnostics, Ba Xaar 'Switzerland', Branched-chain DNA (BayerTM Diagnostics, Emeryville, USA) and NASBA (〇rganon TeknikaTM, Boxtel, Netherlands). However, the above-mentioned goods have the following disadvantages: (丨) operation is time consuming, the steps are cumbersome; (2) the linear range of the detection is not good; and (3) there is no sample internal quality control. For the sake of the job, developing a hepatitis B detection technology that is easy to operate, highly sensitive, and highly reliable is required by the industry. SUMMARY OF THE INVENTION One object of the present invention is to provide a primer for quantifying hepatitis B virus, which can amplify hepatitis B virus DNA, the primer being selected from the group consisting of the following primers and variants thereof: An upstream primer having one of sequence identification number indicating sequences, an upstream primer having a sequence as shown in sequence identification number 3, an upstream primer having a sequence as shown in sequence identification number 4, and having a sequence as shown in sequence identification number 2 One of the /, the heart, the downstream primer of J, has a downstream primer such as one of the sequences of the sequence identification code 5, and a downstream primer having one of the sequences as shown by the sequence identification number 。. Another object of the present invention is A kit for quantifying a hepatitis B virus, comprising a primer as described above. A further object of the present invention is to provide a method for quantifying hepatitis A virus, such as The primer promotes the instant polymerase chain reaction. Detailed Description of the Invention 96645.doc 1262947 The present invention relates to an introduction for quantifying hepatitis B virus, which can be applied Various instant polymerase chain reactions, and thereby quantify hepatitis B virus. The primer according to the present invention can amplify the hepatitis B virus round eight, the primer C: selected from the group consisting of the following primers and variants thereof: having a sequence Identification number 1: the sequence-upstream primer, the upstream primer having one of the sequences as shown in sequence identification number 3, the upstream primer having one of the sequences as shown in sequence identification number 4, and one of the sequences as shown in sequence identification number 2. a downstream primer, a downstream primer having one of the sequences as shown in SEQ ID NO: 5, and a downstream primer having a sequence as shown in SEQ ID NO: 6. Preferably, the primers according to the present invention form the following primer pair: (a) An upstream primer of one of the sequences indicated by the sequence identification number 1 and a downstream primer having a sequence of the sequence identification number 2, (b) an upstream primer having a sequence as shown in the sequence identification number 3, and having a sequence identification number of 5 a downstream primer of one of the sequences shown; and (c) having an upstream primer of one of the sequences shown in SEQ ID NO: 4 and having a sequence as shown in SEQ ID NO: 6. A downstream primer. The "polymerase chain reaction" as used herein includes four steps: (1) denaturation of a template to form two single strands; (7) two primers and two strands of step (1) Adhesing; (3) extending the primers with DNA polymerase, and (4) obtaining two pairs of DNA. The above steps are repeated, and amplification can be obtained for a specific DNA fragment. "Instant Polymerase Chain Reaction" The second is the use of fluorescent detection. The "polymerase chain reaction" occurs to quantify the amount of template involved in the reaction. The current development of the real-time poly-enzyme chain reaction is in a closed reaction tube, in addition to the primer and the base of the polymerase coupled 96645.doc 1262947 lock reaction - and the fluorescent signal is added to the reaction tube The & meter is a cycle-by-cycle measurement, which can achieve the function of simultaneous amplification and simultaneous measurement of signals. Therefore, the instant polymerase chain reaction has the advantages of direct amplification and no post-treatment, which not only saves time and effort, but also avoids post-treatment and treatment. At the same time, because of the cycle-by-cycle η· glory signal, and at the same time depicting the complete pattern of gene duplication, it has better sensitivity and reproducibility. The concentration range of the reagent of the present invention can reach a maximum of (8) to 1〇8, which is the largest of the current methods. The term "variant" refers to a nucleotide molecule that can be substituted for the primer of the present invention and can still amplify the sequence of the same specific fragment, and the lower the temperature; in other words, the primer and the amplification Sequence variation between templates Due to the nature of the polymerase chain reaction itself, even if there is variability in the sequence between the primer and the template to be amplified, a specific DNA fragment can be synthesized by adjusting the reaction temperature of the adhesion step in the polymerization-lock reaction. In other words, the greater the sequence variability between the primer and the template to be amplified, the smaller the reactivity of the adhesion step, the higher the reaction temperature of the adhesion step. Therefore, the person of ordinary knowledge in this particular field according to the present invention It is revealed that different primers can be designed according to the DNA fragment to be amplified, any base substitution, addition or reduction for the primer of the present invention, and the primers which can still amplify the specific fragment with the primer of the present invention are all protected by the present invention. The primer according to the present invention can be applied to a 即时-type instant polymerase chain reaction, for example, can be used for DNA-binding fluorophore (DN A-binding flU〇r〇Ph〇reS), adjacent linear 〇lig〇pr〇bes, 5 nuclease oliclease 〇lig〇pr〇bes, hairpin recruitment probe 96645 .doc 1262947 (hairpin oligoprobes) and self-fluorescing amplicons, etc. The invention further provides a kit of hepatitis B virus comprising the above-described primer or primer pair. In a specific embodiment, the TaqMan® instant polymerase chain reaction system is used. The system uses a target probe to quantify the polymerase chain reaction product. The target probe has its own at 5 and 3, respectively. Two fluorescent groups of the reporter and the quencher. The fluorescent base is the fluorescent portion of the reported protein. The commonly used fluorescent base is FAM. If the target probe is not hybridized with the template, it is located at the 3' end. The quencher fluorophore (usually a long wavelength color, such as red) reduces the reporter fluorophore at the 5' end by Fluorescence Res() nanoeergy transfer (FRET) (usually Fluorescent light of a short wavelength, such as green. When the target probe is denatured to the template and adhered to the single-strand template, 'the polymerase can add the nucleotide acid according to the sequence of the template'. The target probe is removed from the 5th end, thus making the 3ι end quencher The glory base is separated from the reporter at the 5' end, and the reporter can diverge its fluorescent energy. For example, the more the number of polymerase chain reactions occurs, the more the reporter fluoresces, so the measurement report The amount of fluorescent agent can be used to quantify the product of the polymerase chain reaction. Therefore, the kit according to the present invention further comprises a target probe which can be combined with the primer or primer pair according to the present invention. The hepatitis B virus 亀 fragment is heterozygous, and its 5, and 3, respectively, have a _first reporter and a first quencher. The first-reporter-deuteration agent used in the present invention may be a conventional fluorescent group such as a pharmaceutically acceptable agent. Preferably, when a (4) primer pair is used, the target probe has the sequence shown in SEQ ID NO: 96645.doc 1262947 Identification Number 7 or its complementary strand. The signal obtained by the measurement is the actual experimental condition or the operation: 乍: poor, the kit according to the invention further comprises an internal control mechanism, which comprises an internal control plastid having an insert, the The insert comprises: a fragment having no similarity with hepatitis B DNA or a human genome sequence, a DNA fragment amplified by a primer or a primer pair according to the present invention; and an internal control probe, The inner control plastid is heterozygous, the 5th and 3rd ends respectively have a second reporter and a second=; and the second reporter is different from the first-reporter. The instant polymerase chain reaction between the control plastid and the (IV) control probe during operation is performed simultaneously with the sample and the target probe for easy observation. The first reporter and the second quencher used in the present invention may be conventional fluorescent groups, such as VK: as a parent-reporter. Preferably, the insert fragment has a sequence as shown in SEQ ID NO: 12 or its complementary strand, and the internal control probe has a sequence as shown in SEQ ID NO: 10 or its complementary strand. In another embodiment of the invention, a hybrid probe immediate polymerase chain reaction system is used. The system quantifies the polymerase chain reaction product by means of a certain wrong probe and an inductive probe. The U-terminal of the sensing probe has a donor chelation, and the 5th end of the fixed-tin probe has an acceptor-fluorescent light. The body glory can be stimulated by the wavelength of the field, and can transfer energy to the receptor fluorescence, while the stimulated receptor light can emanate - longer wavelengths. If the error-producing probe and the salt-sensing probe are not hybridized with the template, under the excitation of the appropriate wavelength, the donor body can be dissipated, but because it is not adjacent to the receptor glory, the energy cannot be transferred, so it can only be observed. The amount of fluorescent light. If the error-producing probe and the sensing probe are denatured and adhered to the single-strand template in the mold 96645.doc 10 1262947, the donor fluorescence can transfer the energy to the acceptor fluorescence, so the receptor firefly can be observed. The amount of light, such as the number of times the polymerase chain reaction occurs, the more products are used as templates, the more the fluorescence of the donor is transferred to the receptor fluorescence, and the fluorescence of the receptor is observed to be divergent. The product of the polymerase chain reaction can be quantified by measuring the amount of receptor fluorescence. Therefore, the kit according to the present invention further comprises a sensing probe and a certain wrong probe, wherein the third end of the sensing probe has a first donor fluorescent light, and the 5' end of the fixed anchor probe has a first a receptor fluorescent, and the sensing probe and anchoring probe can be hybridized with the amplified hepatitis B virus DNA fragment according to the primer or primer of the present invention, respectively, and when the anchor probe and the sensing probe are Upon simultaneous hybridization to the amplified hepatitis B disease fragment according to the primer or primer pair of the present invention, the energy of the first donor fluorescent light can be shifted to the first acceptor fluorescence. The first acceptor fluorescent light and the first donor fluorescent light used in the present invention may be conventional fluorescent groups, such as using FL as the first donor fluorescent light, and using [C Red^仂 as the first-receptor Fluorescent. Preferably, when the (8) or (4) primer pair is used, the 忒-sensing probe has a column as shown in SEQ ID NO: 8 or its complementary strand, and the anchor probe has, for example, or sequence identification number 9 or its complementary strand In the present embodiment, the specific embodiment also includes a g σ丨 丨 mechanism, one of the internal control plastids of 苴勹a α and an internal control anchor probe, and the dragon 5•戚= a second receptor fluoresce The internal control mass can be interposed with the internal control mass when the internal anchor probe and the inductive probe are ... and the time can be used to shift the internal control mass to the internal (4) And sensory:: ie = 96645.doc 1262947 Synthase chain anti-cologne σ 叩 叩 and 疋 anchor probe and sense for easy observation. It is better to know that the h h wood is known to I 』, and the ^ # 11 i? JL ^ part control anchor probe has a sequence as shown by sequence identification, flat oil or its complementary strands. Preferably, according to the kit of the present invention, the other is used as a quasi-curve + & quasi-shell body, and the standard plastid has an additional fragment slave contained by the invention according to the invention ^ ^ ^ ^ ^ ^ ^ ^ 毒 Poison dna ... ... 对 对 对 对 对 对 对 对 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎 肝炎a sequence as shown in the preferred embodiment. A nucleic acid sequence having a sequence such as the sequence identification number 13 or its complementary vector and all of the "hepatitis" sequences is confirmed. The kit according to the present invention can identify existing known genes. Type, : avoids the occurrence of false detection rate due to differences in viral genotypes or species. The invention is further related to a method for quantifying hepatitis B virus, which is based on the primer of the present invention or The kit is carried out ie, the synthase chain reaction. In other words, when using the TaqMan® instant polymerase chain reaction system, the method according to the invention comprises the following steps: (1) mixing with a target probe at different concentrations of the standard plastid Reaction, the standard The body has an extra fragment comprising a hepatitis B virus DNA fragment amplified by a primer or primer pair according to the present invention, and the target probe is amplified with the pair of primers or primers according to the present invention. The hepatitis B virus DNA fragment is heterozygous, and the 5, and 31 ends of the target probe respectively have - the first reporter - a first 泮 96645.doc 1262947 quencher; (II) the different concentrations in the step (1) The first reported in the plastid reaction is 1 to obtain a standard curve of the standard plastid concentration versus the first reported dose; (III) B derived from the sample of hepatitis B virus; preferably, the sample source From the blood, such as serum or plasma; (·), scorpion or primer pair, target probe, and DM A in the step (out) for immediate polymerase chain reaction; () in the first step (lv) The intensity of the reporter is compared to the standard curve passed in step (ϋ) to quantify the concentration of DNA in the sample. Preferably, the method according to the invention comprises an internal control step by adding an internal control mass to - internal control probe and hepatitis B The virus and the target probe are subjected to steps (iv) and (7), and the amount of the second reporter is measured, and the positive signal is detected, indicating that the reaction condition is successful. In detail, when the (four) probe is instant polymerase chained In the reaction system, the method according to the present invention comprises the following steps: (1) performing heterozygous reaction with a sensing probe and a certain wrong probe at different concentrations of the standard plastid; the standard plastid has an extra fragment, and the additional fragment comprises The amplified hepatitis B virus DNA fragment according to the primer or the primer of the present invention; the 3, end of the sensing probe has a first donor fluorescent light, and the 5th end of the error detecting probe has a first receptor Camping light, the sensing probe and the error-correcting probe can be hybridized with the amplified Hepatitis 3 virus and the eight-segment, respectively, according to the method of the present invention, and the combination is 疋ί "wood needle and induction probe simultaneously Hybridization to the amplified hepatitis B virus DNA fragment according to the introduction of the 96645.doc 1262947 or the primer pair according to the invention, the energy of the first conjugate fluorescence can be transferred to the first receptor fluorescence; (11) Measurement of the standard plastid reaction at different concentrations in step (1) The amount of receptor fluorescence is obtained to obtain a standard curve of the standard plastid concentration versus the amount of receptor fluorescence; (Hi) to obtain DNA derived from the sample of hepatitis B virus; preferably, the sample is derived from blood, such as serum Or plasma; (iv) an instant polymerase chain reaction with DNA in the primer or primer pair, the sensing probe, the deciphering probe, and the step (1) in accordance with the present invention; 00 measuring the receptor fluorescence in step (iv) The intensity is compared to the standard curve obtained in step (9) to quantify the concentration of DNA in the sample. Preferably, the method according to the present invention comprises an internal control step of adding an internal control mass and an internal control anchor probe, and performing steps (4) and (7) with the hepatitis B tear and the anchor probe, and Measuring the amount of light in the second receptor camp, such as producing a positive domain, indicates that the reaction conditions are successful. In the present invention, the extraction of viral sputum in the sample, the immediate polymerization enzyme chain reaction and the analysis of the signal are achieved by a method well known to those skilled in the art. In the preferred sample, the extraction and instant polymerization of viral DNA are carried out. The enzyme chain reaction and signal analysis are illustrated in the examples below. The invention is illustrated by the following examples, which are not intended to be construed as limiting the invention. [Examples] Example 1· Preparation of hepatitis B DNA Preparation of serum samples: 96645.doc -14· 1262947 - The blood of the patient is collected using a blood pump containing no anti-rotation agent, and the blood is collected for about 5 minutes. Blood coagulation; - Centrifuge at 3000 rpm for 10 minutes (complete in room temperature for two hours) - Pipette the upper serum - dispense 50 μί in a microcentrifuge tube
-保存於-20°C B 型肝炎 DNA 卒取步驟（viogene®，Blood & Tissue Genomic Mini，GG1002): -取50 pL血清 -加入5 pL蛋白酶K -加入50 pL Ex緩衝液 -震盪5秒再離心5秒 -於60°C加熱20分鐘 -於70°C加熱20分鐘 -震盪5秒再離心5秒 -加入60 gL異丙醇 -震盪5秒再離心5秒 -於70°C加熱20分鐘 -震盪5秒再離心5秒 -吸取全部溶液至新離心管之内管中（内管+外管） -離心1 3，000 rpm 2分鐘 -倒去外層廢液（外管） -加入130 μί清洗緩衝液 -離心1 3，000 rpm 2分鐘 96645.doc -15- 1262947 -倒去外層廢液（外管） 加入1 3 0 pL清洗緩衝液 -離心13,000 rpm 2分鐘 -丟棄外管層並換新外管 -加入50 μί 70°C無菌水 -離心1 3，000 rpm 2分鐘 -置於4°C立即檢測或儲存於_2〇°C 實例二·· TaqMan®即時聚合酶連鎖反應 將B型肝炎DNA 1 pL與已配製好之聚合酶連鎖反應試劑 24 pL放入96孔盤中，再將96孔盤放入ABI® 7900機器中， 利用水解探針（hydrolysis pr〇be)在進行聚合酶連鎖反應複 製的同時，即時偵測螢光值而定量。其步驟詳述如下： 步驟1.取一管標準品（1〇ig隻病毒/微升）或是以圖i所示之 標準質體作10倍序列稀釋（1〇8、1〇6、1〇4、1〇2、1〇1、1〇〇 病毒/微升）；- Store at -20 °C Hepatitis B DNA Stroke Procedure (viogene®, Blood & Tissue Genomic Mini, GG1002): - Take 50 pL of serum - add 5 pL of proteinase K - add 50 pL of Ex buffer - shake for 5 seconds Centrifuge for 5 seconds - heat at 60 ° C for 20 minutes - heat at 70 ° C for 20 minutes - shake for 5 seconds and then centrifuge for 5 seconds - add 60 g of isopropyl alcohol - shake for 5 seconds and then centrifuge for 5 seconds - heat at 70 ° C for 20 minutes - Shake for 5 seconds and centrifuge for 5 seconds - Pipette all the solution into the inner tube of the new centrifuge tube (inner tube + outer tube) - Centrifuge at 1 3,000 rpm for 2 minutes - Pour off the outer waste (outer tube) - Add 130 μί Wash Buffer - Centrifuge 1 3,000 rpm 2 minutes 96645.doc -15- 1262947 - Pour off the outer waste (outer tube) Add 1 30 pL Wash Buffer - Centrifuge 13,000 rpm for 2 minutes - Discard the outer tube layer and change New outer tube - add 50 μί 70 ° C sterile water - centrifugation 1 3,000 rpm for 2 minutes - immediately at 4 ° C or stored at _2 ° ° C Example 2 · TaqMan® Instant Polymerase Chain Reaction B Hepatitis DNA 1 pL and the prepared polymerase chain reaction reagent 24 pL were placed in a 96-well plate, and the 96-well plate was placed in an ABI® 7900 machine using a hydrolysis probe (h). Ydrolysis pr〇be) Quantifies the fluorescence value while detecting the PCR reaction. The steps are detailed as follows: Step 1. Take a tube of standard (1 〇 ig virus / microliter) or a 10-fold serial dilution of the standard plastid shown in Figure i (1〇8, 1〇6, 1 〇4,1〇2,1〇1,1〇〇virus/μl);
步驟2·準備試劑及耗材，PCR試劑為ABI TaqMan⑧pcRStep 2·Prepare reagents and consumables, PCR reagent is ABI TaqMan8pcR
Core Reagents Kit，Cat. No. N808-0228 ;取 13.125 |uL無菌 水、3·5 μΐ^ 25 mM MgCl2、2.5 μι A緩衝液、各〇·25 吣 1〇 福 dATP/dUTP/dGTP/dCTP、〇·125 卟酵素、〇·25 μίυΝ(}、i 5 μί 10 μΜ具有如序列辨識編號丨所示序列之上游引子、^ $ pL 10 μΜ具有如序列辨識編號2所示序列之下游引子、〇 $ 1〇 μΜ 5’端具有FAM螢光基且具有如序列辨識編號7所 示序列之目標探針及丨pLDNA混合於96孔反應槽中；一併 以圖2所示内部控制質體及5，端具有VIC螢光基且具有如序 96645.doc 1262947 列辨識編號10之内部控制探針進行内部品管偵測； / 驟 3_ 貼上光學黏附蓋（Optical adhesive cover)Pan ^ 43 U 971蓋上均勻加熱板，放置ABI® 7900機器⑷ 井要朝左上角放置）； 。步驟4.上機，其聚合酶連敍應條件㈣。g 2分鐘，％ C 1〇分鐘’再進行5G個循環之95t 15秒、60。(： i分鐘； H成@取數據資料’取出盤（plate)丟棄關掉機器電 源’再關電腦； V驟6.用經序列稀釋已知濃度的標準品，濃度為⑺8至 又病t /U升，根據標準品得〇值數據可畫出標準曲線，其Core Reagents Kit, Cat. No. N808-0228; take 13.125 | uL sterile water, 3·5 μΐ^ 25 mM MgCl2, 2.5 μιη A buffer, each 〇·25 吣1〇福dATP/dUTP/dGTP/dCTP, 〇·125 卟 enzyme, 〇·25 μίυΝ(}, i 5 μί 10 μΜ upstream primer with sequence as shown in sequence identification number 、, ^ $ pL 10 μΜ with downstream primer of sequence shown in sequence identification number 2, 〇 $1〇μΜ The target probe having a FAM fluorophore at the 5' end and having the sequence shown in SEQ ID NO: 7 and 丨pLDNA mixed in a 96-well reaction tank; together with the internal control plastid shown in Figure 2 and 5 The internal control probe with the VIC fluorescent base and the sequence 96645.doc 1262947 column identification number 10 for internal quality inspection; / 3_ attach the optical adhesive cover Pan ^ 43 U 971 cover Heat the plate evenly and place the ABI® 7900 machine (4) with the well facing the upper left corner); Step 4. On the machine, the polymerase is linked to the conditions (4). g 2 minutes, % C 1 〇 minutes' and then 95t 15 seconds, 60 of 5G cycles. (: i minutes; H into @ take the data data 'take out the plate (plate) discard and turn off the machine power 're-close the computer; V step 6. Use the sequence to dilute the known concentration of the standard, the concentration is (7) 8 to the disease t / U liter, according to the standard product devaluation data can draw a standard curve,
、、口果不於圖3 ’且其内部品管摘測圖示於圖4 ;檢體得到Q 值再與標準曲線比對即可得知其濃度，其再現性評估示於 表1 : 曰期 斜率 1 2004/3/23 -3.525 2 2004/4/19 3.607 3 2004/4/22 -3.675 4 2004/4/30 -3.337 5 2004/6/8^ -3.074 平均 -3.443 SD ±0.174 另將本實例之系統與現有市售試劑b_DNA同時偵測“個 檢體，其結果示於圖5 ，兩者相關線性為 R2=0.9472(y=26.116x+70〇〇〇〇)高濃度檢體，為其中b_DNA 只能測出大於5.3X 101G複本數之高濃度檢體，而本實例之 系統則可測出數據，表示其線性範圍優於b-DNA。 實例三：雜合探針即時聚合酶連鎖反應 96645.doc 1262947 將卒取好之B型肝炎DNA 5 和配好之LightCycler®聚 曰轉連鎖反應试劑15 /xL加入玻璃毛細管（Glass capillaries) 中，利用離心混合均勻，放入LightCycler⑧機器進行pcR， 利用雜合探針在聚合酶連鎖反應進行複製的同時，即時偵 測螢光值而定量。 步驟1 ·取一官標準品（丨〇 1 G隻病毒/微升）或是標準質體作 10倍序列稀釋(108、10、104、1〇2、1〇丨、1〇。隻病毒/微升); 步驟2.準備離心轉接器（Centrifuge adapter)、毛細管和試 劑’取6.3 μι無菌水、2.4 卟 25 mM MgCl2、2 〇 吣 1〇 _ 具有如序列辨識編號3所示序列之上游引子、2〇 ι〇 μΜ 具有如序列辨識編號5所示序列之下游引子、〇15叫2〇_ 5’端具有LC Red 640螢光基且具有如序列辨識編號9所示序 列之定錯探針、0.15叫20 μΜ 3,端具FLf光基且具有如序 ，辨識編號8所示序列之感應探針及2叫酵素配製成反應 试劑，一併以含有具有如序列辨識編號12所示序列之内部 控制質體、感應探針及5,端|LCRed7G5螢光基且具有如序 列辨識編號11所示序列之内部控制定㈣針進行内部品管 偵測； 細管放置離心轉接器上依序排 入毛細管，取5 gL稀釋好的標準 列 步驟3.取所需數量的毛 ，取15 /iL配好的試劑加 口口依序加入毛細官，取5/xL的檢體（B型肝炎DNA)接在標 準品之後依序加入毛細管； v驟4.將毛細官蓋上蓋子，經離心则G rpm 3G秒，將毛 細管依序放置轉盤(LC car〇Usel)上，轉盤放入叫抓咖⑧ 96645.doc 1262947 機器； 步驟5·上機，其聚合酶連鎖反應條件為95它1〇分鐘，再 進行50循環95。(： 〇秒、5(rc 1〇秒、72。〇 1〇秒，之後降 溫至4(TC 30秒； 步驟6.完成讀取數據資料，取出轉盤將毛細管丟棄，關 掉機器電源，再關電腦； 步驟7·用經序列稀釋已知濃度的標準品，濃度為1〇8_1〇0 又病I /耄升。根據標準品所得Ct值數據可畫出標準曲線， 其結果示於圖6，且其内部品管偵測圖示於圖7，檢體之α 值再與標準曲線比對即可得知其濃度，其再現性評估示於 表2 : 曰期 ~ 斜率 1 2004/3/18 -3.554 2 2004/3/25 _ -3.564 3 2004/4/1 -3.512 4 2004/4/8 -3.586 5 2004/4/22 -3.513 6 2004/4/29 -3.544 7 2004/5/6 -3.719 8 2004/5/13 -3.615 9 2004/5/20 -3.717 10 2004/5/27 -3.684 平均 -3.601 SD ±0.069 另將本貫例之糸統與現有市售試劑b-DNA同時偵測3 8個 檢體，其結果示於圖8 ，兩者相關線性為 R2=0.8439(y=62.362x+700000)高濃度檢體，b-DNA只能測 出大於5.3 X 1 〇複本數之高濃度檢體，而本實例之系統則 可測出數據，表示其線性範圍優於b-DNA。 96645.doc 1262947 實例四··標準血清與即時聚合酶連鎖反應之對應關係 以世界衛生組織（WHO)訂購之標準血清與根據本發明之 方法定量所得結果之對應關係示於表3，且其換算方程式示 於圖10。 表3 ·· 才示準血清 (IU) 1 ~S數 即時聚合酶連鎖反應 (複本數） 對數 50000 4.69897 6.00E+03 3.778296 12500 4.09691 1.40E+03 Γ 3.147367 3125 3.49485 3.36E+02 2.526339 781.25 2.89279 8.05E+01 1.905634 390.025 2.59176 3.69E+01 1.567497 上述實施例僅為說明本發明之原理及其功效，而非限制 本毛月^於此技術之人士對上述實施例所做之修改及變 化仍不堤背本發明之精神。本發明之權利範圍應如後述之 申晴專利範圍所列。 【圖式簡單說明】 圖1為例示本發明之標準質體構築示意圖。 圖2為例不本發明之内部控制質體構築示意圖。 圖為以TaqMan®即時聚合酶連鎖反應之標準曲線圖。 圖:為以丁aqMan®即時聚合酶連鎖反應之内部品管倘測 之I =x TaqMan⑧即時聚合酶連鎖反應系統與市售試劑 之比較結果圖。 … 圖6為以 „ Ρ夺1合_連鎖反應之標準曲線圖。 圖7為以雜合探針卽拄取人^ 圖 。 卩~ t合酶連鎖反應之内部品管偵測 圖8為以雜合探針g 士 可聚合酶連鎖反應系統與市售試劑 96645.doc -20- 1262947 之比較結果圖。 圖9為以TaqMan®即時聚合酶連鎖反應與雜合探針即時 聚合酶連鎖反應系統之比較結果圖。 圖10為標準血清與即時聚合酶連鎖反應之對應關係換算 方程式圖。 96645.doc •21 - 1262947 序列表 <110> 國立成功大學 <120>定量B型肝炎病毒之引子及引子對套組及方法 <130〉無 <160> 13 <170> Patentln version 3.2 <210〉 1 <211〉 22 <212〉DNA <213> 人工序列 <400〉 1 gcaggtcccc tagaagaaga ac <210〉 2 <211> 17 <212〉DNA <213〉人工序列 <400〉 2 cgacgcggcg attgaga <210〉 3 <211> 19 <212〉DNA <213> 人工序列 <400〉 3 gaccaccaaa tgcccctat <210> 4 <211〉 21 <212〉DNA <213> 人工序列 <400> 4 gaccaccaaa tgcccctatc t <210> 5 <211> 22 <212〉DNA <213〉人工序列 96645.doc 221262947 <400> 5 cgagattgag atcttctgcg ac <210> 6 <211> 22 <212> DNA <213> 人工序列 <400> 6 ggagattgag atcttctgcg ac <210〉 7 <211> 17 <212> DNA <213〉人工序列 <400〉 7 cctcgcctcg cagacga <210〉 8 <211〉 24 <212> DNA <213〉人工序列 <220〉 <221〉 變異區 <222> (3)..(3) <223> s>4gic <400〉 8 gasgcaggtc ccctagaaga agaa <210〉 9 <211〉 26 <212〉DNA <213〉人工序列 <220> <221〉 變異區 <222〉（19)..(19) <223> m為 a 或c <220> <221> 變異區 <222〉（22)..(22) <223〉r 為 a或 g <400> 9 tccctcgcct cgcagacgma grtctc 22 17The results are not shown in Figure 3' and the internal quality control chart is shown in Figure 4. The Q value of the sample is compared with the standard curve to know its concentration. The reproducibility evaluation is shown in Table 1: Period slope 1 2004/3/23 -3.525 2 2004/4/19 3.607 3 2004/4/22 -3.675 4 2004/4/30 -3.337 5 2004/6/8^ -3.074 Average - 3.443 SD ±0.174 The system of the present example simultaneously detects "a sample" with the existing commercially available reagent b_DNA, and the results are shown in Fig. 5. The linear correlation between the two is R2=0.9472 (y=26.116x+70〇〇〇〇) high concentration sample. For the high-concentration samples in which b_DNA can only detect more than 5.3X 101G copies, the system of this example can measure the data, indicating that its linear range is better than b-DNA. Example 3: Hybrid probe instant polymerase Chain reaction 96645.doc 1262947 Add the well-prepared hepatitis B DNA 5 and the prepared LightCycler® polyfluorene chain reaction reagent 15 /xL to a glass capillaries, mix it by centrifugation and place it in a LightCycler8 machine. Perform pcR, using hybrid probes to replicate in the polymerase chain reaction, and instantly detect fluorescence values. Step 1. Take an official standard (丨〇1 G virus/μl) or a standard plastid for 10-fold serial dilution (108, 10, 104, 1〇2, 1〇丨, 1〇. Virus/μl); Step 2. Prepare Centrifuge adapter, Capillary and Reagent 'take 6.3 μl sterile water, 2.4 卟25 mM MgCl2, 2 〇吣1〇_ with sequence as shown in Sequence Identification Number 3 The upstream primer, 2〇ι〇μΜ has a downstream primer as shown in sequence identification number 5, and the 〇15 is called 2〇_ 5' end has a LC Red 640 fluorescent base and has a sequence as shown in sequence identification number 9. The wrong probe, 0.15 is called 20 μΜ 3, the end is FLf-based and has the order, the sensing probe of the sequence number No. 8 and the 2 called enzyme are formulated into reagents, together with the sequence identification number The internal control mass, the sensing probe and the 5, end | LCRed7G5 fluorescent base of the sequence shown in Figure 12 have an internal control set (4) for sequence identification as shown in sequence identification number 11 for internal quality inspection; The capillary is sequentially discharged into the capillary, and 5 gL of the diluted standard column is taken. Step 3. Take the required amount. For the hair, take 15 / iL with the reagent and mouth to add the capillaries sequentially, take 5 / xL of the specimen (hepatitis B DNA) after the standard is added to the capillary; v. 4. Will the capillary officer Cover the lid, centrifuge for G rpm for 3G seconds, place the capillary on the carousel (LC car〇Usel), turn it into the machine called the graffiti 8 96645.doc 1262947; Step 5 · On the machine, its polymerase chain reaction The condition is 95 for 1 minute, and then 50 cycles for 95. (: leap second, 5 (rc 1 sec, 72. 〇 1 〇 second, then cool down to 4 (TC 30 sec; Step 6. Finish reading data, remove the turntable to discard the capillary, turn off the machine power, then turn off Computer 7; Step 7: Dilute the standard concentration of the standard with a concentration of 1〇8_1〇0 and I/耄. According to the Ct value data obtained from the standard, a standard curve can be drawn. The result is shown in Fig. 6. And the internal quality control detection diagram is shown in Figure 7. The alpha value of the sample is compared with the standard curve to know its concentration. The reproducibility evaluation is shown in Table 2: 曰期~ slope 1 2004/3/18 -3.554 2 2004/3/25 _ -3.564 3 2004/4/1 -3.512 4 2004/4/8 -3.586 5 2004/4/22 -3.513 6 2004/4/29 -3.544 7 2004/5/6 - 3.719 8 2004/5/13 -3.615 9 2004/5/20 -3.717 10 2004/5/27 -3.684 Average - 3.601 SD ±0.069 Also use this method to simultaneously detect b-DNA with existing commercially available reagents 3 8 samples, the results are shown in Figure 8, the correlation linearity is R2 = 0.8439 (y = 62.362x + 700,000) high concentration of the sample, b-DNA can only measure more than 5.3 X 1 〇 the number of copies Concentration sample, while the system of this example is measurable The data indicates that its linear range is superior to b-DNA. 96645.doc 1262947 Example 4 · Correspondence between standard serum and immediate polymerase chain reaction The standard serum ordered by the World Health Organization (WHO) and the method according to the present invention are quantified. The corresponding relationship between the results obtained is shown in Table 3, and the conversion equation is shown in Fig. 10. Table 3 ·· shows the quasi-serum (IU) 1 ~ S number instant polymerase chain reaction (replica number) logarithm 50000 4.69897 6.00E+03 3.778296 12500 4.09691 1.40E+03 Γ 3.147367 3125 3.49485 3.36E+02 2.526339 781.25 2.89279 8.05E+01 1.905634 390.025 2.59176 3.69E+01 1.567497 The above examples are only illustrative of the principles and effects of the present invention, and are not intended to limit this month. The modifications and variations of the above-described embodiments of the present invention are not intended to be exhaustive. The scope of the present invention should be as set forth in the scope of the patents of the present disclosure. [Simplified Description of the Drawings] FIG. Schematic diagram of the standard structure of the present invention. Fig. 2 is a schematic view showing the construction of the internal control mass of the present invention. The figure is a standard curve of the TaqMan® instant polymerase chain reaction. Figure: A comparison of the I = x TaqMan8 instant polymerase chain reaction system with commercially available reagents for the internal quality control of the Dingaq®® Instant Polymerase Chain Reaction. Fig. 6 is a standard curve of „ 1 1 _ chain reaction. Fig. 7 is a diagram of a human probe with a hybrid probe. Figure 8 shows the internal quality control of the 卩~ t synthase chain reaction. Comparison of the results of the hybrid probe g-s-polymerase chain reaction system and the commercially available reagent 96645.doc -20- 1262947. Figure 9 shows the instant polymerase chain reaction system with TaqMan® instant polymerase chain reaction and hybrid probe Comparison of the results. Figure 10 is a conversion equation for the correspondence between standard serum and real-time polymerase chain reaction. 96645.doc •21 - 1262947 Sequence Listing <110> National Cheng Kung University <120> Quantification of Hepatitis B Virus And the primer pair method and method <130>none<160> 13 <170> Patentln version 3.2 <210> 1 <211> 22 <212>DNA <213> artificial sequence <400> 1 Gcaggtcccc tagaagaaga ac <210> 2 <211> 17 <212>DNA <213>Artificial sequence <400> 2 cgacgcggcg attgaga <210> 3 <211> 19 <212>DNA <213> Artificial sequence <400> 3 gaccaccaaa tgcccctat <210> 4 <211 21 <212>DNA<213> artificial sequence <400> 4 gaccaccaaa tgcccctatc t <210> 5 <211> 22 <212>DNA <213> artificial sequence 96645.doc 221262947 <400> Cgagattgag atcttctgcg ac <210> 6 <211> 22 <212> DNA <213> Artificial sequence <400> 6 ggagattgag atcttctgcg ac <210> 7 <211> 17 <212> DNA <213 〉Artifical sequence <400〉 7 cctcgcctcg cagacga <210> 8 <211> 24 <212> DNA <213>Artificial sequence <220>221> Variant zone <222> (3).. (3) <223>s>4gic<400> 8 gasgcaggtc ccctagaaga agaa <210> 9 <211> 26 <212>DNA <213>artificial sequence <220><221>;222>(19)..(19)<223> m is a or c <220><221> Variation region <222>(22)..(22) <223>r is a or g <400> 9 tccctcgcct cgcagacgma grtctc 22 17
24twenty four
96645.doc -2- 26 1262947 <210〉 10 <211> 17 <212> DNA <213> 人工序列 <400> 10 ctcttgctga agcttct 17 <210> 11 <211〉 25 <212> DNA <213> 人工序列 <400> 11 cttgctgaag cttctgacta cgact 25 <210〉 12 <211> 190 <212〉DNA <213> 人工序列 <400〉 12 tttggtgtct ttcggagtgt ggattcgcac tcctcctgct tacagaccac aaaatgcccc 60 tatcttatca acacttccgg aaactgctgt tgttagacga cgaggcaggt cccctagaag 120 aagaactctt gctgaagctt ctgactacga ctaatcgccg cgtcgcagaa gatctcaatc 180 tcgggaatct 190 <210〉 13 <211> 190 <212> DNA <213> 人工序列 <400> 13 tttggtgtct ttcggagtgt ggattcgcac tcctcctgct tacagaccac aaaatgcccc 60 tatcttatca acacttccgg aaactgctgt tgttagacga cgaggcaggt cccctagaag 120 aagaactccc tcgcctcgca gacgaaggtc tcaatcaccg cgtcgcagaa gatctcaatc 180 tcgggaatct 190 96645.doc96645.doc -2- 26 1262947 <210> 10 <211> 17 <212> DNA <213> Artificial sequence <400> 10 ctcttgctga agcttct 17 <210> 11 <211> 25 <212> DNA <213> Artificial sequence <400> 11 cttgctgaag cttctgacta cgact 25 <210> 12 <211> 190 <212>DNA <213> Artificial sequence <400> 12 tttggtgtct ttcggagtgt ggattcgcac tcctcctgct tacagaccac aaaatgcccc 60 tatcttatca acacttccgg aaactgctgt tgttagacga cgaggcaggt cccctagaag 120 aagaactctt gctgaagctt ctgactacga ctaatcgccg cgtcgcagaa gatctcaatc 180 tcgggaatct 190 < 210> 13 < 211 > 190 < 212 > DNA < 213 > artificial sequence < 400 > 13 tttggtgtct ttcggagtgt ggattcgcac tcctcctgct tacagaccac aaaatgcccc 60 tatcttatca Acacttccgg aaactgctgt tgttagacga cgaggcaggt cccctagaag 120 aagaactccc tcgcctcgca gacgaaggtc tcaatcaccg cgtcgcagaa gatctcaatc 180 tcgggaatct 190 96645.doc