臺灣博碩士論文加值系統

迴路恆溫增幅反應(loop-mediated isothermal amplification, LAMP) 由Notomi等人所發展，係利用4-6個特異性引子辨識並結合至目標核酸序列，反覆地進行引子延展與置換作用，於短時間內大量增幅核酸片段。本研究期以此技術發展出快速比色法(rapid colorimetric assay)與即時雙重迴路恆溫增幅反應分析法(real-time duplex LAMP, dLAMP)來偵測樣本中的病毒核酸，供血液篩檢、臨床診斷與監控、流行病學調查等之應用。
快速比色法係以人類巨細胞病毒UL97基因序列為偵測目標，HCMV-LAMP反應條件經最適化(Optimization)與重複測試，每個反應可偵測的最低複本數為30。而呈色原理則利用溶液中鹽離子濃度，會引發金奈米粒子不等程度之聚集；當鹽分越高，金奈米粒子聚集越明顯而溶液顏色將由紅色轉變為藍色。於HCMV-LAMP反應完成後加入膠體金溶液呈色，可觀察到溶液顏色隨增幅產物濃度遞增而呈現藍、紫、紅等不同色澤。以影像記錄、紫外光/可見光分光光譜儀測量雙波長(OD650 / 520)動力學變化，可知焦磷酸鎂與DNA產物的數量會減緩金奈米粒子聚集速度。本研究亦嘗試建立即時雙重迴路恆溫增幅反應，於單一試管內同步偵測B型肝炎病毒(HBV)與C型肝炎病毒(HCV)核酸。經重複測試，每個反應可偵測的HBV最低複本數為50、HCV則為60。該增幅產物經解離曲線分析，可知HBV與HCV二者解離溫度(melting temperature, Tm)不同，且再現性良好；與限制酶切割試驗(BspMI與Tth111I)結果相符。進一步分析臺灣地區常見的HBV基因型及HCV亞型病毒株等dLAMP增幅產物之Tm值，以建立信賴區間(confidence interval) 做為鑑別HBV及HCV之依據；其中HBV的信賴區間為85.49 ± 1.34°C、HCV則為88.90 ± 0.81°C。後續模擬臨床實驗室之檢驗流程，自國立臺灣大學附設醫院收集已確診HBV或HCV感染者之臨床檢體進行dLAMP與信賴區間之測試。結果顯示dLAMP陽性預測率為88 %，而Tm值信賴區間之鑑別正確率為88.64 %；其中HBV鑑別正確率為83.72 %、HCV 鑑別正確率為93.33 %。
快速比色法的特異性及靈敏度與現有的比濁法相近，增幅效果主要取決於HCMV-LAMP引子對之設計優劣。以化學還原法合成膠體金溶液，不僅成本低廉，保存也相當容易。採取肉眼觀察顏色變化，適合用於快速篩檢。而雙重迴路恆溫增幅反應，除了即時偵測樣本中病毒核酸，其增幅產物經解離溫度分析亦可快速地鑑別HBV與HCV。但dLAMP的偵測靈敏度與正確率(尤其是HBV的偵測)不夠理想，還需要諸多努力以改善其效能。

Loop-mediated isothermal amplification (LAMP) was established by Notomi et al. (2000). It uses 4-6 specific primers to recognize the target gene sequence and rapidly amplify under isothermal condition. Using this technique, we established a rapid colorimetric assay and a real-time duplex LAMP (dLAMP) method for viral nucleic acid detection. We anticipated that these two assays could be applied in blood screening, clinical diagnosis, and epidemiological investigation in the future.
In the rapid colorimetric assay, we first optimized and validated the HCMV-LAMP reaction, the viral detection limit was about 30 copies per reaction. Furthermore, we mixed colloidal gold with LAMP products. Gold nanoparticles (AuNPs) became accumulated and solution color changed from red to blue quickly in HCMV-LAMP negative situation. But in positive situation, the solution color kept in red more longer. This phenomenon might be based on decreasing the ionic strength along with increasing DNA numbers of the solution after the HCMV-LAMP reaction. For the dLAMP method, HBV and HCV were detected simultaneously. The viral detection limits were 50 copies for HBV and 60 copies for HCV. In viral identification, we first collected clinical samples containing HBV or HCV strains of different genotypes or subtypes, respectively. By post-dLAMP melting curve analysis, HBV and HCV had different and non-overlapped Tm confidence intervals as 85.49±1.34°C and 88.90±0.81°C, respectively. We also tested the possibility of clinical application of the dLAMP and Tm analysis in detection and differentiation of HBV and HCV. The positive predictive value (PPV) of dLAMP method was 88 %. The accuracy of Tm differentiation of HBV and HCV was 88.64 %, that of HBV was 83.72 %, HCV 93.33 %.
In conclusion, the specificity and sensitivity of colorimetric assay were similar to those of turbidimetry. Reading colors by naked-eye was simple and easy. This end-point assay is suitable for rapid qualitative analysis. However, the duplex LAMP detected the viral nucleic acid in real-time manner and differentiated HBV and HCV conveniently by melting temperature analysis. But the efficiency and accuracy rate were not satisfactory, and further improvement is required.

口試委員審定書 (i)
中文摘要 (ii)
英文摘要 (iv)

第一章 緒論(1)
第一節 人類巨細胞病毒(HCMV)(1)
一、病毒基本構造(1)
二、病毒複製週期(1)
三、傳播方式與流行病學(2)
四、臨床病徵(3)
五、治療藥物(3)
六、實驗室診斷(4)
第二節 肝炎病毒(hepatitis viruses)(5)
一、B型肝炎病毒(5)
二、C型肝炎病毒(5)
三、臨床病徵(6)
四、臨床預防與治療藥物(6)
五、實驗室診斷(7)
第三節 迴路恆溫增幅反應(7)
一、反應試劑(8)
二、增幅反應(8)
三、增幅產物偵測方法(9)
第四節 金奈米粒子簡介(10)
第五節 研究動機與目的(10)

第二章 材料與方法(11)
第一節 細胞與病毒培養(11)
一、細胞培養(11)
二、病毒培養(11)
第二節 核酸萃取與確認(12)
一、核酸萃取(12)
二、聚合酶連鎖反應(13)
三、洋菜膠電泳(14)
第三節 質體標準品製作(14)
一、增幅目標序列(15)
二、轉型作用(16)
三、質體萃取(17)
四、限制酶切割試驗(17)
五、核酸定序(18)
第四節 HCMV迴路恆溫增幅反應(20)
一、設計LAMP引子對(20)
二、反應條件最適化(21)
三、偵測靈敏度與特異性(22)
第五節 金奈米粒子比色法(22)
一、金奈米粒子合成(22)
二、快速比色法(23)
第六節 雙重迴路恆溫增幅反應(duplex LAMP)(24)
一、統合反應條件(24)
二、解離曲線分析(24)
三、限制酶切割試驗(25)
四、臨床樣本收集與處理(25)
五、統計分析(26)

第三章 結果(27)
第一節 快速比色法之建立(27)
一、病毒培養與核酸萃取(27)
二、質體標準品之建立(27)
三、HCMV-LAMP(28)
四、合成金奈米粒子與呈色(29)
第二節 雙重迴路恆溫增幅反應(30)
一、反應條件最適化(30)
二、解離溫度預測與分析(31)
三、限制酶切割試驗(31)
四、建立Tm值信賴區間(31)
五、臨床檢體測試(32)

第四章 討論(33)
參考文獻(37)
圖表(48)
附錄(76)

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