臺灣博碩士論文加值系統

本論文目的為建置可同時偵測四種血清型登革熱病毒且具有高靈敏性、高特異性之血液篩檢系統，並實際篩檢高雄市登革熱高峰期之捐血族群，以評估登革熱於高雄市流行期間對輸血安全性的影響。TaqMan Real-time RT-PCR可以快速偵測到血漿中少量的登革熱病毒核酸，檢測靈敏性每個反應可達0.99~20 TCID50、重複性測試其變異係數< 4％及特異性達100％；並於檢測過程加入內控制組，控管檢測品質。利用此核酸篩檢系統檢驗登革熱最高峰時期9,618位捐血人及良心回電發燒血漿133例，檢測結果並無發現陽性案例。另外，採用商用化Dengue NS1 Ag試劑套組，篩檢登革熱高峰期1,703位捐血人血漿及發燒捐血人血漿133例，結果1例陽性、 22例未確認；陽性案例經RT-PCR確認後結果為陰性反應。因Dengue NS1 Ag檢測未確認之比例高達1 %，推論此方法並不適用於捐血人登革熱篩檢。台灣血液基金會為提升血液品質回饋於社會大眾，建置登革熱篩檢系統以備登革熱疫情擴大時隨時可啟動篩檢機制。本研究檢測樣本數為抽樣篩檢，但依初步結果推論在台灣地區登革熱並不會造成輸血安全的影響。

The purpose of this study was to develop the high sensitive and specific screening method to detect for four serotypes dengue viruses. Use this method to assay the blood donor group in Kaohsiung city during the peak of dengue fever to evaluate the blood safety. TaqMan Real-time RT-PCR can be rapid screening the low concentration nucleic acid of dengue virus in serum. The sensitive is 0.99 ~ 20 TCID50 per reaction, coefficient of repeatability variance < 4% and specific is 100% in the system. There is an internal control in detect process to monitor the test quality. Samples were collected from 9,618 donors during the peak of dengue fever and 133 recall donors who is fever. Serum samples were detected by TaqMan Real-time RT-PCR and no one is positive. In addition, we utilized Dengue NS1 Ag commercial kits to screen 1,703 donors’ serum during the peak of dengue fever and 133 donors who is fever. The results showed that only one case is positive and 22 equivocal. The reactive sample was confirmed by RT-PCR and the result is negative. The equivocal rate is 1 % of Dengue NS1 Ag commercial kits, so the kit is not applicable for dengue virus screening in blood donors. To enhance blood quality, Taiwan Blood Service Foundation (TBSF) developed the rapid screening method of dengue viruses to start screening program when the dengue fever breakout. According our data indicate
there is no effect of dengue fever in transfusion security.

【目錄】
目錄-----------------------------------------------------------I

表目錄--------------------------------------------------------Ⅵ
圖目錄--------------------------------------------------------Ⅶ
附錄----------------------------------------------------------Ⅷ
中文摘要------------------------------------------------------Ⅸ
英文摘要------------------------------------------------------Ⅹ
前言-----------------------------------------------------------1
一、研究背景-------------------------------------------------1
二、登革熱之流行病學-----------------------------------------2
三、登革熱病徵之臨床分類-------------------------------------3
四、登革熱病毒之傳播與分類-----------------------------------5
五、登革熱病毒之實驗室診斷-----------------------------------6
(一)登革熱病毒分離--------------------------------------6
(二)血清學抗體檢測試驗----------------------------------7
(三)登革熱抗原檢測試驗----------------------------------7
(四)登革熱病毒核酸檢測----------------------------------8
六、本研究應用之分析方法 ------------------------------------9
(一) TaqMan 即時定量 RT-PCR-----------------------------9
(二) 登革熱NS1抗原酵素免疫分析法-----------------------9
七、研究目的------------------------------------------------10
研究架構------------------------------------------------------12
材料與方法----------------------------------------------------13
一、實驗材料------------------------------------------------13
(一)細胞及病毒培養藥品---------------------------------13
(二)生物材料-------------------------------------------14
(三)試劑套組-------------------------------------------15
(四)實驗用之引子對及探針序列---------------------------16
(五)儀器設備-------------------------------------------17
二、自動化儀器系統之選用------------------------------------18
(一)自動化混樣儀器-------------------------------------18
(二)自動化核酸萃取儀器---------------------------------18
(三)Real-Time PCR 儀器----------------------------------18
(四)DENGUE NS1 AG半自動化檢測系統-----------------------19
三、實驗方法----------------------------------------------20
(一)C6/36細胞株培養------------------------------------20
(二)四種血清型之登革熱病毒培養-------------------------20
(三)病毒培養液收集-------------------------------------21
1.重複凍融法----------------------------------------21
2.玻璃磨砂棒均質法----------------------------------21
(四)50 %病毒感染力價------------------------------------22
(五)TaqMan Real-time RT-PCR方法建立---------------------24
1.引子及探針比對設計與評估--------------------------24
2.神經壞死病毒(內控制組)培養------------------------24
3.設定反應條件--------------------------------------25
四、樣本檢測------------------------------------------------26
(一)登革熱高峰期一般捐血人血漿-------------------------26
(二)發燒良心回電之捐血人保留血漿-----------------------26
結果----------------------------------------------------------27
一、TaqMan Real-Time RT-PCR方法建立--------------------------27
(一)登革熱病毒之培養放大-------------------------------27
1.C6/36細胞株之培養---------------------------------27
2.四種血清型登革熱病毒培養之細胞病變觀察------------27
3.50 %病毒感染力價----------------------------------28
(二) 引子對及探針測試----------------------------------28
(三) 內控制組測試與CT允收值設定------------------------28
二、One-step TaqMan Real-time RT-PCR方法之確效---------------29
(一) 靈敏性--------------------------------------------29
1.血清型第一型登革熱病毒檢測靈敏性------------------30
2.血清型第二型登革熱病毒檢測靈敏性------------------30
3.血清型第三型登革熱病毒檢測靈敏性------------------30
4.血清型第四型登革熱病毒檢測靈敏性------------------31
(二) 重複性--------------------------------------------31
(三) 特異性--------------------------------------------32
三、TaqMan Real-time RT-PCR樣本檢測結果----------------------33
(一) 一般捐血人血漿檢測--------------------------------33
(二) 良心回電發燒之保留血漿檢測 -----------------------33
四、PLATELIATM DENGUE NS1 AG試劑套組樣本檢測結果--------------33
(一) 一般捐血人血漿檢測--------------------------------33
(二) 良心回電發燒之保留血漿檢測 -----------------------34
討論----------------------------------------------------------34
參考文獻------------------------------------------------------38
表------------------------------------------------------------41
圖------------------------------------------------------------65
附錄----------------------------------------------------------87















【表目錄】
表一、比對四種血清型登革熱高度保留區之3’UTR核酸序列----------41
表二、四種血清型登革熱病毒之TCID50定量結果----------------------42
表三、彙整內部控制組之CT值測試結果-----------------------------46
表四、血清型第Ⅰ型登革熱病毒培養液檢測靈敏性-------------------47
表五、血清型第Ⅱ型登革熱病毒培養液檢測靈敏性-------------------50
表六、血清型第Ⅲ型登革熱病毒培養液檢測靈敏性-------------------53
表七、血清型第Ⅳ型登革熱病毒培養液檢測靈敏性-------------------56
表八、登革熱TaqMan Real Time RT-PCR重複性測試-------------------59
表九、TaqMan Real-time RT-PCR樣本檢測結果-----------------------61
表十、PLATELIATM DENGUE NS1 AG試劑套組樣本檢測結果----------------62
表十一、推估2010年高雄市登革熱無症狀感染機率-------------------63






【圖目錄】
圖一、C6/36細胞株培養型態--------------------------------------65
圖二、連續觀察各種血清型登革熱病毒培養於 C6/36細胞株，所造成之
細胞病變效應-------------------------------------------66
圖三、不同引子對之靈敏性測試-----------------------------------69
圖四、一般血漿PCR反應之內控制組CT值分佈圖----------------------71
圖五、添加不同濃度之第Ⅰ型登革熱病毒PCR反應之內控制組CT值分佈圖--------------------------------------------------------72
圖六、添加不同濃度之第Ⅱ型登革熱病毒PCR反應之內控制組CT值分佈圖--------------------------------------------------------73
圖七、添加不同濃度之第Ⅲ型登革熱病毒PCR反應之內控制組CT值分佈圖--------------------------------------------------------74
圖八、添加不同濃度之第Ⅳ型登革熱病毒PCR反應之內控制組CT值分佈圖--------------------------------------------------------75
圖九、血清型第Ⅰ型登革熱病毒培養液檢測靈敏性之Z計分質量控制圖--------------------------------------------------------76
圖十、血清型第Ⅱ型登革熱病毒培養液檢測靈敏性之Z計分質量控制圖--------------------------------------------------------78
圖十一、血清型第Ⅲ型登革熱病毒培養液檢測靈敏性之Z計分質量控制圖------------------------------------------------------80
圖十二、血清型第Ⅳ型登革熱病毒培養液檢測靈敏性之Z計分質量控制圖------------------------------------------------------82
圖十三、特異性干擾測試-----------------------------------------84
圖十四、陰性檢體特異性測試-------------------------------------85












【附錄】
附表一、PLATELIATM DENGUE NS1 AG 靈敏性及特異性分析-------------87
附圖一、疾病管制局全國2008~2010年登革熱疫情分析趨勢圖----------88
附圖二、磁珠病毒核酸萃取試劑套組編號202之核酸萃取流程----------90
附圖三、Labturbo自動化混樣系統 --------------------------------91
附圖四、自動化磁珠核酸萃取儀-----------------------------------92
附圖五、ABI StepOne即時定量PCR儀器-----------------------------93
附圖六、TECAN RMP 150全自動化酵素免疫分析儀---------------------94
附圖七、BIO-TEK MQX200全波長酵素免疫分析儀----------------------95
附錄一、研究計劃審查同意書-------------------------------------96

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