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研究生：	李怡臻 Lee, Yi-Chen
論文名稱：	藉由即時聚合酶連鎖反應附帶熔解曲線區分B型肝炎病毒在抗病毒藥物治療下的病毒變異株 Discrimination of hepatitis B virus mutants under antiviral treatment by real-time PCR with melting curve analysis
指導教授：	楊孔嘉 Young, Kung-Chia 張定宗 Chang, Ting-Tsung
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2008
畢業學年度：	96
語文別：	中文
論文頁數：	92
中文關鍵詞：	抗藥性、熔解曲線分析法、B型肝炎病毒
外文關鍵詞：	HBV, anti-drug, melting curve analysis
相關次數：	點閱：116 下載：1
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目前全球將近有三億五千萬人口屬於慢性B型肝炎感染患者，長期感染B型肝炎病毒(HBV)的情況下，患者可能演變其他嚴重的肝臟疾病，例如肝硬化或是肝癌。現在核准用於治療HBV感染的藥物主要是口服核酸類似物，包括：干安能(lamivudine；LAM)、干適能(adefovir dipivoxil；ADV)、貝樂克(entecavir；ETV) 與喜必福(telbivudine；LdT) 這四種，這些藥物會和HBV的反轉錄酶/DNA聚合酶結合，進而抑制HBV的DNA複製，達到治療效果。但是在長期的藥物治療下，很容易產生抗藥性突變株而使藥物失去治療能力，這些抗藥性突變株主要是在HBV反轉錄酶/DNA聚合酶位置發生突變使得藥物無法和其結合，現在已知這些藥物常見產生抗藥性的位點分別為對LAM產生抗性的L180M 與 M204I/V，對ADV產生抗藥性的A181V、I233V與N236T，對ETV產生抗性的T184G、S202I與M250V以及對LdT產生抗藥性的M204I，其中ETV的抗藥性位點必須同時伴隨有L180M與M204I/V。因此，我們針對這些抗藥性位點設計了MCA 640、LA+E 705、ADV 705三組雜交探針(hybridization probe)進行突變株的偵測，先以即時聚合酶連鎖反應(Real-Time PCR)進行定量後，再以熔解曲線法將不同的突變株進行區分。在定量的結果，此三組探針在103-1011 copies/ml之間維持良好線性關係，且可以明確偵測到103 copies/ml的HBV DNA，三組探針之間的一致性也很高，更增加其定量結果的可信度。在區分突變株的熔解曲線法分析中，LA＋E 705探針藉由熔解曲線可以明顯的區分出三種病毒株，包含不具抗藥性的野生株、帶有M204I/V而對LAM產生抗藥性的突變株以及同時有M204I/V和S202I位點突變而對ETV與LAM產生抗藥性的突變株；而MCA 640探針則區分出帶有A181V而對ADV產生抗藥性突變株，以及同時有L180M和T184G而對ETV產生抗藥性的變異株。熔解曲線法雖然容易受到HBV本身其他非抗藥性位點的突變而影響判讀，但是卻比其他方式敏感度高，且可同時進行定量，具有應用於臨床上的可看性。

Nearly 350 million people are chronically infected with hepatitis B virus (HBV) worldwide. After prolonged infection, patients frequently develop severe liver diseases such as liver cirrhosis and hepatocellular carcinoma. Nucleoside or nucleotide analogues are used to treat HBV infected patients including lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), and telbivudine (LdT), which mainly act by inhibition of HBV polymerase activity resulting in decrease of viral replication. However, long-term therapy is commonly associated with the emergence of resistant mutants. Eight major mutations are observed in the polymerase gene during using these antiviral drugs. There are L180M and M204I/V for LAM, 204I for LdT, A181V, I233V, and N236T for ADV, and T184G, S202I, and M250V for ETV. Interestingly, the ETV resistance positions in addition to preexisting LAM-resistance mutations M204I/V and L180M are necessary for viral resistance to ETV. We designed three sets of hybridization probes which were MCA 640, LA+E 705 and ADV 705 to quantify HBV DNA by real-time PCR and discriminate these mutations resistant to antiviral drugs by melting curve simultaneously. All of three hybridization probe sets have a well linear regression in range from 103 to 1011 copies/ml and have a good sensitivity of 103 copies/ml. The hybridization probe, LA+E 705, could discriminate three HBV strains including wild type, M204 I/V, or combined M204I/V with S202I by melting curve analysis clearly. The mutation of M204 indicates resistance to LAM, and the combination of M204I/V with S202I is associated with resistance to both LAM and ETV. The MCA 640 probe could be used to detect A181V mutants resistant to ADV in all genotypes, and L180M mutants combined with T184G mutation, which were resistant to ETV and LAM. Although the temperature of melting curve analysis was interfered by HBV quasispecies, this method with high sensitivity for low copies and simultaneous HBV DNA quantification was better than other methods. This experimental protocol and methological designs reported herein will be extensively favorable for application in clinical diagnosis.

中文摘要-------------------------------------------------------------------Ⅰ
英文摘要-------------------------------------------------------------------Ⅲ
目錄-------------------------------------------------------------------------Ⅳ
誌謝-------------------------------------------------------------------------Ⅴ
表 / 圖 / 附錄 目錄----------------------------------------------------Ⅸ
壹、序論 (Introduction) ------------------------------------------------ 1
一.	B型肝炎病毒 Hepatitis B virus (HBV) ----------------------------2
1.	B型肝炎病毒發現史-------------------------------------------2
2.	B型肝炎病毒的流行病學--------------------------------------2
3.	B型肝炎病毒的基因體與基本構造---------------------------3
4.	B型肝炎病毒的生活史----------------------------------------4
5.	B型肝炎病毒的症狀與自然病程-----------------------------4
6.	B型肝炎病毒的分型------------------------------------------6
二.	治療B型肝炎病毒的藥物-----------------------------------------7
1.	干擾素治療-----------------------------------------------------7
2.	口服抗病毒藥物------------------------------------------------8
三.	B型肝炎病毒的抗藥性---------------------------------------------10
四.	B型肝炎病毒抗藥性病毒株的分型--------------------------------12
貳、目的與策略（Aim & Strategy）--------------------------------14
參、材料與方法 (Materials and Methods) --------------------------15
一.	病人檢體收集-----------------------------------------------------17
二.	B型肝炎病毒去氧核糖核酸萃取--------------------------------17 
三.	B型肝炎病毒反轉錄酶基因片段的選殖------------------------18
三. 1放大B型肝炎病毒反轉錄酶基因片段的聚合酶連鎖反應18
        三. 2膠體電泳---------------------------------------------------- 19
        三. 3聚合酶連鎖反應產物的純化------------------------------- 20
        三. 4 T Easy載體黏合反應---------------------------------------21
        三. 5大腸桿菌轉型-----------------------------------------------22
        三. 6細菌冷凍液製備-------------------------------------------- 25
        三. 7小量質體萃取-----------------------------------------------25
        三. 8 DNA定序準備工作-----------------------------------------26
        三. 9中量質體萃取-----------------------------------------------27
四.	構築各種B型肝炎病毒抗藥性位點質體------------------------29
        四. 1 定點突變—PCR-drived overlap extension ----------------29
        四. 2 定點突變-- QuikChangeⅡSite-Directed Mutagenesis Kit-32
五.	即時聚合酶反應附帶熔解曲線分析----------------------------- 34
        五. 1配製各個構築質體標準液----------------------------------34
        五. 2即時聚合酶連鎖反應附帶溶解曲線分析------------------34
六.	臨床檢體定序-----------------------------------------------------40
        六. 1 聚合酶連鎖反應------------------------------------------- 40
        六. 2 聚合酶連鎖反應產物的純化------------------------------ 41
        六. 3構築含檢體RT片段的質體進行分析--------------------- 41
        六. 4外送定序準備----------------------------------------------42
七.	限制酶切割片段多樣性分析-------------------------------------42
肆、結果 (Results)------------------------------------------------------- 46
一.	設計聚合酶連鎖反應附帶熔解曲線分析法的探針與引子------47
二.	以聚合酶連鎖反應附帶熔解曲線分析法區分突變株-----------48
1.	依據Tm值的不同區分突變株--------------------------------48
2.	熔解曲線分析法最低偵測極限------------------------------- 49
3.	混合型突變株的偵測與比例極限-----------------------------50 
4.	熔解曲線分析法的再現性-------------------------------------50
5.	臨床檢體分析--------------------------------------------------51
三.	Real-Time PCR的定量--------------------------------------------53
四.	直接定序臨床檢體確認突變位置--------------------------------53
五.	限制酶切割片段多樣性(RFLP)分析檢體M204I/V突變-------- 53
六.	熔解曲線法的準確度與錯誤------------------------------------- 54
七.	針對特定檢體挑選single colony分析單一病毒株的序列-------55
伍、討論 (Discussion )--------------------------------------------------57
一.	實驗設計上的困難-------------------------------------------- 57
二.	不同方法比較探討-------------------------------------------- 58
三.	熔解曲線法的臨床判讀與應用之展望----------------------- 60
陸、參考文獻 (References )------------------------------------------- 63
表/圖 (Tables and Figures)
附錄 (Appendixes)



表 / 圖 / 附錄 目錄
表一、三組探針上含各種抗藥性位點質體所對應的Tm值緩衝範圍----67
表二、LA＋E 705探針的三重複試驗，顯示熔解曲線法再現性高--------68
表三、三組探針用於檢體分析的Tm值及判讀-----------------------------69
表四、相同臨床檢體以不同組探針進行定量結果-------------------------70
表五、17個臨床檢體直接定序結果----------------------------------------71
表六、10個檢體以臨床RFLP方式分析結果-------------------------------72
表七、C02125檢體15株單一病毒序列分析--------------------------------73
圖一、三組偵測突變病毒株的探針與其相配合引子的位置示意圖------74
圖二、MCA 640探針的熔解曲線示意圖-----------------------------------75
圖三、LA＋E 705探針的熔解曲線示意圖---------------------------------76
圖四、ADV 705探針的熔解曲線示意圖-----------------------------------77
圖五、三組探針熔解曲線分析偵測的最低濃度----------------------------78
圖六、LA＋E 705探針用於混合感染的熔解曲線分析--------------------79
圖七、MCA 640探針的臨床檢體分析結果--------------------------------80
圖八、LA＋E 705探針的臨床檢體分析結果-------------------------------81
圖九、ADV 705探針的臨床檢體分析結果-------------------------------82
圖十、三組探針用於定量的標準曲線--------------------------------------83
圖十一、兩兩比較三組探針定量相同檢體的一致性----------------------84
圖十二、臨床用檢驗M204突變點的方法---------------------------------85
圖十三、熔解曲線法區分抗藥性病毒株判讀準則-------------------------86
附錄一、HBV生活史-------------------------------------------------------87
附錄二、口服核酸類似物的結構示意圖-----------------------------------88
附錄三、定點突變所用引子—PCR-drived overlap extension--------------89
附錄四、附錄四、定點突變所用引子-- By QuikChange® Site-Directed Mutagenesis Kit Primers------------------------------------------90
附錄五、各個抗藥性病毒株質體標準品的序列---------------------------91
附錄六、熔解曲線分析法的原理-------------------------------------------92
                                    

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校內：2009-07-24公開
校外：2011-07-24公開

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