臺灣博碩士論文加值系統

本研究的目的是評估EasyChip® HPV Blot應用於子宮頸抹片樣本之人類乳突瘤病毒DNA基因定型之效能、效度與信度，當進行大規模社區HPV篩檢時也導入品質保證系統以確保HPV基因定型結果的品質。以分析靈敏度、分析特異性、靈敏度評估HPV Blot的效能。以39種HPV基因型質體評估HPV Blot的分析靈敏度與分析特異性，以細胞株及子宮頸檢體評估靈敏度。用於評估的39種HPV基因型質體(HPV 6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7, and MM8)含有HPV基因組之L1基因片段。每一個質體視為一個基因組相等物。HPV Blot之效度與信度以臨床試驗評估，試驗的黃金標準是20個L1型別專一性PCR。以靈敏度、特異性與準確性評估效度，McNemar’s Test與Kappa統計量評估信度。評估的方式分別針對受試者與個別基因型。品質保證系統包括操作環境的維持、HPV Blot可靠度與再現性的評估及資料判讀。品質保證系統包含品質保證標準，包括操作時7個外部控制組及HPV Blot基因定型時所需的3個內部控制組。每一批次實驗包含89個子宮頸檢體與7個外部控制組。7個外部控制組由Caski細胞株、HeLa細胞株、Jurkat細胞株、男性血液細胞DNA、無菌水及一對配對檢體所組成。而配對檢體是源自相同樣本的分裝液。Caski細胞株、HeLa細胞株、Jurkat細胞株及配對檢體用於評估包括DNA抽取、PCR及HPV基因定型等所有的操作。男性血液細胞DNA與無菌水只用於PCR和HPV基因定型的操作。本研究需要100批次實驗進行分析。Jurkat細胞株與男性血液細胞DNA可當作操作環境中有無潛在污染的外源HPV DNA的指標，而無菌水可當作操作環境中有無潛在污染的外源DNA的指標。
HPV Blot分析靈敏度的偵測極限：HPV 16是1個基因組相等物；HPV 6、26、31、33、37、39、43、44、45、54、55、61、67、68、69、82、CP8061、CP8304、L1AE5、MM7及MM8是10個基因組相等物；HPV 11、18、32、35、42、51、52、53、56、58、59、66、70、74及MM4是20個基因組相等物；HPV 62及72是50個基因組相等物。每一樣本其HPV檢測的分析靈敏度是1~50基因組相等物的拷貝數。HPV Blot的分析特異性是39種辨識不同基因型HPV之個別探針不會與其他基因型HPV的PCR產物相互雜交，無交叉反應。在細胞株檢驗的靈敏度：Caski細胞DNA分別以膠體電泳及HPV Blot檢測的靈敏度為10-3與10-4 ng DNA；HeLa細胞DNA分別以膠體電泳及HPV Blot檢測的靈敏度為10-2與10-3 ng DNA。靈敏度的計算是參照Caski細胞的基因組含有約60~600拷貝的HPV 16基因組，HeLa細胞的基因組含有約10~50拷貝的HPV 18基因組。因此，以細胞株DNA為模板則HPV Blot之檢測極限：HPV 16與HPV 18的靈敏度分別是1~10與2~8的拷貝數。HPV Blot對子宮頸細胞檢體中HPV 16與HPV 18病毒之檢測極限為10個拷貝數。
HPV Blot對受試者的敏感度、特異性與準確性分別為97% (95%CI：94.0% – 98.0%)、96%(95%CI：92.0% – 98.0%)及96.5% (95%CI：94.0%-98.0%)。MY11/GP6+ - HPV Blot對受試者的McNemar’s Test、Kappa統計量與P值分別為0.25、0.93(95%CI：0.89-0.96) 及0.62，顯示HPV Blot與黃金標準的比較是優良的一致性。HPV Blot對個別基因型的敏感度是100%有HPV 6, 11, 31, 35, 45, 59及66；95~99%有HPV 16, 18, 33及58；90~94%有HPV 51, 52, 53及70；80~89%有HPV 39, 56及68；低於79%有HPV 62及CP8061。HPV Blot對個別基因型的特異性是100%有HPV 6, 11, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 62, 66, 68, 70及CP8061；95~99%有HPV 16。HPV Blot對個別基因型的準確性是100%有HPV 6, 11, 35, 45, 59及66；95~99%有HPV 16, 18, 31, 33, 39, 51, 52, 53, 56, 58, 62, 68, 70及CP8061。HPV Blot對個別基因型的一致性是完美的(Kappa=1)有HPV 6, 11, 35, 45及59；優良的(0.99 < Kappa < 0.81)有HPV 16, 18, 31, 33, 39, 51, 52, 53, 58, 66, 68, 70, 62及56；令人滿意的(0.8 < Kappa < 0.61)有HPV CP8061。
所有的實驗均嚴格遵守品質保證系統的四項規範。因此，100批次實驗中每一批次其3個HPV陰性外部控制組的結果都是HPV陰性，表示沒有HPV偽陽性的結果。Caski、HeLa及Jurket細胞株在2000個細胞量的規模中對於HPV 16、HPV 18及GAPDH的基因定型結果有100%的靈敏度與特異性。5個外部控制組的結果符合品質保證系統規範的需求，顯示HPV Blot在操作上有優良的可靠度。批次內與批次間的再現性分別為98%與97%。HPV Blot之基因定型結果需先判斷其三個內部控制組為合格，其基因定型結果才能確認。本研究之基因定型鑑定結果的品質得以保證。
HPV Blot用於HPV感染之臨床與流行病學的研究是一種高靈敏度的、準確的、可靠的及可再現的工具。本研究對於HPV基因定型導入品質保證系統，它包含7個外部控制組與3個內部控制組，對於實驗與實驗室操作有適當的規範並證明有效。由本研究的發現證實了HPV Blot對於臨床之HPV基因定型、專一性型別HPV持續感染的追蹤及專一性型別HPV疫苗的開發有很大的價值。

The aim of this study was to evaluate the performance, validity and reliability of the EasyChip® HPV Blot for Human papillomavirus (HPV) DNA genotyping in cervical swab samples, and assured the quality of HPV typing in large-scale community-based cervical neoplasia screening by conducting a quality assurance system. The performance of HPV Blot was assessed by analytic sensitivity、analytic specificity and sensitivity. The analytic sensitivity and specificity of HPV Blot was assessed by using 39 HPV genotype plasmids, and assessed sensitivity by using cell lines and cervical specimens, respectively. The 39 types of HPV (HPV 6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7, and MM8) were assessed using plasmids DNA containing L1 region of HPV genome. Each plasmid was considered as a genome equivalent (geq). A clinical trial was conducted to assess the validity and reliability of HPV Blot. The gold standard of trial was 20 L1 type-specific parallel PCR. The assessment of validity was sensitivity, specificity and accuracy. The assessment of reliability was McNemar’s Test and Kappa statistic. The way of assessment was by subject and by type, respectively. The quality assurance system includes the maintenance of operating environment, assessment of reliability, assessment of reproducibility, and blot interpretation. The quality assurance standards include the assessment of seven extrinsic controls in operation and three intrinsic controls of HPV Blot in genotypes identification. Each batch experiment contained samples from 89 cervical specimens and seven extrinsic controls. The seven extrinsic controls were Caski cells, HeLa cells, Jurkat cells, male human blood cell DNA, sterile water, and paired sibling controls. The replicated aliquots of the same sample were a pair of sibling controls. Caski cells, HeLa cells, Jurkat cells and sibling controls were used simultaneously to assess entire procedures inclusive of DNA extraction, PCR, and HPV typing. Male human blood cell DNA and sterile water were used only in the assessment of PCR and HPV typing. One hundred batch experiments were conducted. Jurkat cells and male human blood DNA were used as an index of potential contamination with exogenous HPV sequences. Sterile water was used as an index of potential contamination with any exogenous DNA sequences.
The detection limits for analytical sensitivities of HPV Blot are 1 geq per PCR for HPV 16, 10 geq per PCR for HPV 6, 26, 31, 33, 37, 39, 43, 44, 45, 54, 55, 61, 67, 68, 69, 82, CP8061, CP8304, L1AE5, MM7 and MM8, 20 geq per PCR for HPV 11, 18, 32, 35, 42, 51, 52, 53, 56, 58, 59, 66, 70, 74 and MM4, and 50 geq per PCR for HPV 62 and 72. The overall analytical sensitivity of HPV detection is 1 to 50 copies of HPV geq for each sample. The analytic specificity of the HPV Blot is each of 39 HPV genotype probes that had no cross-reactivity with amplicons of other HPV genotypes. In cell lines, the detection limits for Caski and HeLa cells were 10–4 and 10–3 ng DNA, respectively, by the HPV Blot and 10–3 and 10–2 ng DNA, respectively, by the gel. The calculated copies are 1~10 for HPV 16 and 2~8 for HPV 18. Both copy numbers were expected on the basis of known HPV 16 and HPV 18 copy numbers in Caski cells (60–600 copies per cell) and HeLa cells (10–50 copies per cell). About 10 copies of HPV 16 and 18 viruses within cervical specimens were detected.
The sensitivity, specificity and accuracy of HPV Blot by subject are 97, 96 and 96.5%, respectively. The McNemar’s Test、Kappa and P value of HPV Blot by subject are 0.25, 0.93 and 0.62, respectively. The sensitivity, specificity and accuracy of HPV Blot by type are 90, 92 and 90.6%, respectively. The McNemar’s Test、Kappa and P value of HPV Blot by type are 2.81, 0.81 and 0.10, respectively. The comparison of HPV Blot and gold standard suggests that is excellent agreement. The sensitivity of HPV Blot by type is 100% for HPV 6, 11, 31, 35, 45, 59 and 66, 99~95% for HPV 16, 18, 33 and 58, 94~90% for HPV 51, 52, 53 and 70, 89~80% for HPV 39, 56 and 68, and less than 79% for HPV 62 and CP8061. The specificity of HPV Blot by type is 100% for HPV 6, 11, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 62, 66, 68, 70 and CP8061, and 99~95% for HPV 16. The accuracy of HPV Blot by type is 100% for HPV 6, 11, 35, 45, 59 and 66, and 99~95% for HPV 16, 18, 31, 33, 39, 51, 52, 53, 56, 58, 62, 68, 70 and CP8061. The type-specific agreement of HPV Blot is perfect (Kappa=1) for HPV 6, 11, 35, 45 and 59, excellent (0.99 < Kappa < 0.81) for HPV 16, 18, 31, 33, 39, 51, 52, 53, 58, 66, 68, 70, 62 and 56, good (0.8 < Kappa < 0.61) for HPV CP8061.
All experiments obeyed 4 criteria of quality assurance system as follows. Thus, all three HPV-negative extrinsic controls were HPV negative in 100 batch experiments. In other words, there was no HPV false-positive result. The HPV typing results for Caski, HeLa, and Jurkat cells indicated that the HPV Blot had 100% sensitivity and specificity for HPV 16, HPV 18, and GAPDH at a scale of 2000 cells. That five extrinsic controls met the requirements of quality assurance system in 100 batch experiments indicate that the HPV Blot had excellent reliability in operation. Intra-batch and inter-batch reproducibility of the HPV Blot were 98% and 97%, respectively. Reader identified three intrinsic controls of HPV Blot, so typing results of HPV Blot were confirmed. In the study, the qualities of identification were qualified.
In conclusion, the HPV Blot is a highly sensitive, accurate, reliable and reproducible tool for clinical and epidemiological studies on HPV infection. This study is conducted a quality assurance system comprising seven extrinsic controls and three intrinsic controls for HPV typing, which has been validated as an appropriate criterion for experiments and laboratory operation. These findings confirm that the HPV Blot is of great value for clinical HPV typing, follow-up of type-specific HPV persistence and development of type-specific HPV vaccines.

口試委員會審定書 第 1 頁
謝辭 第 2 頁
圖目錄 第 7 頁
表目錄 第 8 頁
中文摘要 第 9 頁
英文摘要 第11頁
第一章 研究背景與動機 第13頁
第二章 文獻回顧 第14頁
2.1人類乳突瘤病毒感染與子宮頸癌 第14頁
2.2人類乳突瘤病毒基因定型的重要性與臨床使用 第15頁
2.3 HPV的檢驗方法與發展 第16頁
2.3.1實驗室常用的檢測技術 第16頁
2.3.2現行發展的檢測與鑑定HPV基因型之體外診斷試劑 第17頁
2.3.2.1 HPV基因定型體外診斷試劑 第17頁
2.3.2.2檢測HPV感染試劑(無法分型) 第18頁
2.4 人類乳突瘤病毒感染的預防 第19頁
2.5 EasyChipÒ HPV Blot的技術原理與規格 第19頁
2.5.1 HPV Blot分析與操作原理 第19頁
2.5.2 HPV Blot規格表 第20頁
2.5.3 HPV基因定型的探針配置 第21頁
2.6 HPV基因定型檢驗的品質保證 第21頁
2.7 體外診斷試劑的效度與信度評估 第22頁
第三章 研究目的、研究設計與統計分析 第26頁
3.1人類乳突瘤病毒基因定型點墨的效能評估 第26頁
3.1.1研究目的 第26頁
3.1.2 研究設計 第26頁
3.1.2.1 研究材料 第26頁
3.1.2.1.1 DNA抽取 第26頁
3.1.2.1.2質體DNA 第26頁
3.1.2.1.2.1 分析靈敏度的評估 第26頁
3.1.2.1.2.2 分析特異性的評估 第27頁
3.1.2.1.3細胞株 第27頁
3.1.2.1.3.1 靈敏度的評估 第27頁
3.1.2.1.4 子宮頸檢體DNA 第27頁
3.1.2.1.4.1靈敏度的評估 第27頁
3.1.2.1.5 男性血液細胞DNA 第27頁
3.1.2.2 研究方法 第28頁
3.1.2.2.1 MY11/GP6+ PCR 第28頁
3.1.2.2.2 HPV Blot基因定型 第28頁
3.1.2.2.2.1雜交流程 第28頁
3.1.2.2.2.2漂洗流程 第28頁
3.1.2.2.2.3 Blocking and Conjugate流程 第29頁
3.1.2.2.2.4呈色流程 第29頁
3.1.3 統計分析 第29頁
3.1.3.1 分析靈敏度的評估 第29頁
3.1.3.2 分析特異性的評估 第29頁
3.2人類乳突瘤病毒基因定型點墨之效度與信度的評估 第30頁
3.2.1研究目的 第30頁
3.2.2 研究設計 第31頁
3.2.2.1試驗目的 第31頁
3.2.2.1.1主要目標 第31頁
3.2.2.1.1.1 檢測檢體被HPV感染之效度與信度的評估 第31頁
3.2.2.1.1.2 鑑定檢體HPV基因定型的效度與信度的評估 第31頁
3.2.2.1.1.3試驗時間 第31頁
3.2.2.2人體試驗委員會與受試者同意書 第31頁
3.2.2.3試驗設計流程 第32頁
3.2.2.4隨機分碼與盲性作業 第33頁
3.2.2.4.1盲性作業程序 第33頁
3.2.2.4.2檢體標示 第33頁
3.2.2.4.3可揭露盲性的標籤 第33頁
3.2.2.4.4 操作程序 第33頁
3.2.2.5所需的檢體樣本數 第34頁
3.2.2.6納入條件的選擇 第34頁
3.2.2.6.1第一階段(400例) 第34頁
3.2.2.6.1.1納入條件 第34頁
3.2.2.6.1.2排除條件 第34頁
3.2.2.6.2第二階段 第35頁
3.2.2.6.2.1納入條件 第35頁
3.2.2.6.2.2排除條件 第35頁
3.2.2.7受試者接受臨床處置之分配方法 第35頁
3.2.2.8研究方法 第36頁
3.2.2.8.1 DNA抽取 第36頁
3.2.2.8.2 GAPDH PCR 第36頁
3.2.2.8.3 MY11/GP6+ PCR 第37頁
3.2.2.8.4 HPV Blot基因定型 第37頁
3.2.2.8.5 HPV L1型別專一性PCR (黃金標準) 第38頁
3.2.2.8.5.1 L1型別專一性PCR的分析靈敏度 第38頁
3.2.2.8.5.1.1質體DNA 第38頁
3.2.2.8.5.1.2分析靈敏度的評估 第38頁
3.2.2.8.5.2 L1型別專一性PCR 第38頁
3.2.2.9數據的管理與品質保證 第39頁
3.2.3統計分析 第39頁
3.2.3.1效度的評估 第39頁
3.2.3.2信度的評估 第39頁
3.3人類乳突瘤病毒基因定型點墨操作的品質保證 第40頁
3.3.1研究目的 第40頁
3.3.2 研究設計 第40頁
3.3.2.1 品質保證系統 第40頁
3.3.2.1.1 操作環境的維持 第40頁
3.3.2.1.2 HPV Blot可靠度的評估 第41頁
3.3.2.1.3 HPV Blot再現性的評估 第41頁
3.3.2.1.4 資料判讀規範 第41頁
3.3.2.2 研究材料 第42頁
3.3.2.2.1 細胞株 第42頁
3.3.2.2.2男性血液細胞DNA 第43頁
3.3.2.2.3 無菌水 第43頁
3.3.2.2.4 配對檢體 第43頁
3.3.2.2.5 子宮頸細胞檢體 第43頁
3.3.2.3 研究方法 第44頁
3.3.2.3.1 實驗品質監測之設計 第44頁
3.3.2.3.2 檢體分裝 第44頁
3.3.2.3.3 DNA抽取 第45頁
3.3.2.3.4 PCR 第46頁
3.3.2.3.4.1 MY11/GP6+ PCR 第46頁
3.3.2.3.4.2 GAPDH PCR 第46頁
3.3.2.3.5 HPV Blot基因定型 第46頁
3.3.2.3.6有效檢測結果之判定原則 第46頁
3.3.2.3.6.1實驗批次有效結果之判定 第46頁
3.3.2.3.6.2有效HPV Blot基因定型結果判讀 第47頁
3.3.2.3.6.2.1人工判讀結果 第47頁
3.3.2.3.6.2.2機器判讀結果 第48頁
3.3.2.3.6.2.3人工和機器判讀結果比對 第48頁
3.3.3 統計分析 第48頁
第四章 研究結果 第49頁
4.1人類乳突瘤病毒基因定型點墨的效能評估 第49頁
4.1.1分析靈敏度與分析特異性 第49頁
4.1.2靈敏度 第49頁
4.1.2.1細胞株 第49頁
4.1.2.2子宮頸檢體 第49頁
4.2人類乳突瘤病毒基因定型點墨的效度與信度評估 第50頁
4.2.1 L1型別專一性PCR之分析靈敏度 第50頁
4.2.2合格與不合格檢體收納的描述性統計 第50頁
4.2.3 抹片判讀結果與病理切片檢查 第50頁
4.2.4型別專一性PCR、MY11/GP6+ PCR、20型及39型HPV Blot檢測之HPV盛行率 第50頁
4.2.5 型別專一性PCR與HPV Blot的一致性比較 第51頁
4.2.6 HPV Blot IC (內部控制組)的效度分析 第52頁
4.2.7型別專一性PCR與HPV Blot所測得之HPV基因型的分布 第52頁
4.2.8型別專一性PCR與HPV Blot對子宮頸組織切片之病理分類的檢出率 第52頁
4.2.9子宮頸組織切片之病理分類其HPV基因型別的分布 第53頁
4.2.10子宮頸組織切片之病理分類其多重感染基因型別的分布 第53頁
4.2.11 MY11/GP6+ PCR與HPV Blot的一致性比較 第54頁
4.3人類乳突瘤病毒基因定型點墨操作的品質保證 第56頁
4.3.1操作環境的維持 第56頁
4.3.2 HPV Blot的可靠度 第56頁
4.3.3 HPV Blot的再現性 第56頁
4.3.4 HPV基因定型資料的判讀 第56頁
第五章 討論 第57頁
5.1人類乳突瘤病毒基因定型點墨的效能評估 第57頁
5.2人類乳突瘤病毒基因定型點墨的效度與信度評估 第58頁
5.3人類乳突瘤病毒基因定型點墨操作的品質保證 第66頁
第六章 結論 第67頁
第七章 研究限制 第69頁
第八章 參考文獻 第70頁
圖與表 第81頁
附錄 第117頁
附錄一、受試者同意書 第117頁
附錄二、臨床記錄表 第121頁
附錄三、檢體代碼說明表 第122頁
附錄四、試驗結果登記總表 第123頁
附錄五、Types Specific PCR檢測結果登記表 第124頁
附錄六、『HPV Blot』檢測結果登記表 第125頁

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