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研究生:尚君璽
論文名稱:利用反轉錄-聚合酵素鏈反應建立病媒蚊帶登革病毒的偵測系統
論文名稱(外文):Establishment of a dengue virus surveillance system in mosquitoes by reverse-transcriptase polymerase chain reaction(RT-PCR)
指導教授:金傳春金傳春引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:流行病學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:109
中文關鍵詞:登革病毒
外文關鍵詞:Dengue fever
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由於登革病毒感染發生率的增加及流行區域的擴大,登革熱已經成為本世紀最重要的蟲媒病毒傳染病。臺灣自從民國70年在小琉球發生登革熱大流行以來,至民國84、85年間臺北縣市也爆發流行,其間幾乎每年均有疫情傳出;近年因為有四型登革病毒同時存在,病媒蚊的密度在某些地區也始終居高不下,致使未來幾年之內發生登革熱/登革出血熱的可能性大幅增加,對國人的生命健康造成感脅。目前臺灣的登革熱偵測仍是以醫師報告的疾病通報系統為主,但此作法多為「被動式」偵測,在防治上的時效性不佳;再加上疑似病例必須經實驗室確診,需時更長。有鑑於此,本研究嘗試將疾病的偵測工作提前至病媒蚊感染登革病毒的階段,建立較為敏感的快速偵測系統。
由於反轉錄─聚合酵素鏈反應(RT-PCR)比傳統的病毒分離法敏感度高,實驗所需時間也較短,因此本研究在方法上是先以胸腔注射登革病毒以得到遭感染的病媒蚊,再採用反轉錄─聚合酵素鏈反應來測定病媒蚊的帶原情形,並比較1.病毒核酸萃取方法、2.使用引子對、3.注射登革病毒之血清型、4.病毒在蚊體內的培養天數、5.病媒蚊種類,以及6.病媒蚊叢聚大小對RT-PCR所造成的影響。最後嘗試根據此實驗結果初步應用在現有田野病媒蚊檢體的檢測工作上,並同時以免疫螢光反應實驗法進行比較。
結果發現:1.利用RNA zol BTM套裝試劑萃取較傳統酚─氯仿萃取法簡便,效果也比Ultraspec ⅢTM理想;2.不論是第一、第二、第三型登革病毒,或是白線、埃及斑蚊,NS3引子對的效果都比C/prM及NS5引子對來得穩定理想;3.第一、第二、第三型登革病毒均可在病媒蚊體內藉RT-PCR測得,但第一型登革病毒僅有NS3可測得,而三組引子對均可測出第二型登革病毒;4.病媒蚊在胸腔注射第一及第二型登革病毒登革病毒後,均在培養不到2天的時間即可偵測出,而第三型登革病毒則需要4-7天的培養;5.埃及斑蚊和白線斑蚊在各項比較因子結果中並無明顯差異;6.病媒蚊叢聚大小(pool size)在實驗階段至少可達40隻。若應用在今(1997)年2月至5月由預研所病昆組所採集的病媒蚊檢體。初步檢驗結果均為陰性,但一週蚊細胞培養後的免疫螢光測試可發現在屏東市豐榮里、臺南市竹溪里及勞工公園等地區捕獲的病媒蚊叢聚有少許登革病毒陽性感染細胞。
本研究結論是在實驗室感染的病媒蚊,其攜帶登革病毒的情形是可以經由病媒蚊胸腔注射及調整反轉錄-聚合酵素鏈反應(RT-PCR)實驗步驟中的相關因子而偵測出來的,但是應用在田野病媒蚊檢體之偵測,仍需要進一步的研究加以改良,以提高其敏感度及特異性,並可同時以免疫螢光測試來確認其結果。未來嘗試將病媒蚊的密度指數用蚊子實際帶病毒的狀況加以修飾,並配合氣象學的資料,以期得到一個更能預測「實際流行可能性」的指標,在流行季節開始之前即以此散佈警訊,及早進行病媒蚊的防治工作,以期能更有效地防治登革熱/登革出血熱的發生。
Dengue fever has become the most important arboviral infectious disease in the 20 century because of the increasing of incidence rates and expanding of the epidemic areas. In 1981, a DEN-2 outbreak exploded in Liu-Chiu Hsiang(琉球鄉). Since then, dengue cases were reported each year in Taiwan. There are four serotypes of dengue viruses and the high indices of mosquito vectors in Taiwan, so that potential risk of DHF/DSS outbreak in the main island for the coming years is increasing. Currently, the surveillance system of dengue in Taiwan depends mainly on physician-based reporting which has weakness in timeliness plus most suspected cases need long time for laboratory confirmation by virus isolation. Therefore, the specific aim of this study was to set up a more sensitive surveillance system to detect dengue virus in mosquito vectors by reverse transcriptase-polymerase chain reaction(RT-PCR).
Since RT-PCR is more sensitive and rapid than traditional virus isolation method, we intrathoracically inoculated dengue viruses into female Aedes to obtain infected mosquitoes and then detected the agent by RT-PCR. The effects of the following 6 parameters in RT-PCR were compared: (1)the viral RNA extraction methods, (2)the primer sets, (3)the serotypes (DEN-1, 2, 3) of inoculated dengue viruses, (4)the duration of the extrinsic period of dengue virus in mosquitoes, (5)the strains of vectors(Aedes aegypti and Aedes albopictus), and (6)pool sizes. In addition, all of above parameters were adjusted and applied to the surveillance on field caught mosquitoes, and then compared these RT-PCR products with those results of indirect immunofluorescence assay(IFA).
The RT-PCR results showed that: (1)RNA zol BTM kit was more efficient than both traditional phenol-chloroform method and Ultraspec IITM kit in RNA extraction of dengue viruses. (2)NS3 primer sets were more stable than both C/prM and NS5 primer sets regardless the serotypes of dengue virus or mosquitoes strains were tested. (3)DEN-1 was always detected only by NS3 primer set, while DEN-2 was detectable by all 3 primer sets(C/prM, NS3, NS5). (4)The intrathoracically inoculated DEN-1 and DEN-2 in Aedes were both detectable by RT-PCR within 2days post-infection whereas DEN-3 was not until 4-7 days post-infection. (5)There was no significant difference in early time detection between Aedes aegypti and Aedes albopictus, and (6)pool size was 40 at least. The preliminary data on the field-caught mosquitoes in Fungrung Li of Pingtung City, Chushi Li and Labor Park of Tainana City collected by National Institute of Preventive Medicine in Taiwan showed negative RT-PCR results but with few(1-2) positive immunofluorescent cells after I week culture in C6/36 cell line.
In conclusion, dengue viruses in mosquito vectors are detectable by RT-PCR plus intrathoracic inoculation in laboratory. Field-caught mosquitoes need further research to improve its sensitivity and specificity. Immunofluorescence assay can be used simultaneously to verify the results of RT-PCR. Future efforts need to improve the current widely used mosquito larvae indices plus virus carrier rate for better prediction of epidemics and prevention of possible DHF/DSS outbreaks.

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