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研究生:黃日芳
研究生(外文):Jih-fang Huang
論文名稱:應用Lig重組蛋白質以酵素結合免疫吸附法進行豬鉤端螺旋體症之血清流行病學調查
論文名稱(外文):Use of recombinant Lig antigen-based enzyme-linked immunosorbent assay for the serological survey of porcine leptospirosis
指導教授:林春福林春福引用關係潘銘正潘銘正引用關係
指導教授(外文):Chuen-fu LinMing-jeng Pan
學位類別:碩士
校院名稱:中臺科技大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:137
中文關鍵詞:顯微凝集試驗Lig重組蛋白質酵素結合免疫吸附法鉤端螺旋體症
外文關鍵詞:LeptospirosisLig proteinmicroscopic agglutination test (MAT)enzyme-linked immunosorbent assay (ELISA)
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鉤端螺旋體症為鉤端螺旋體菌所引起之重要人畜共通傳染疾病,常見於熱帶及亞熱帶地區。可感染各種哺乳類動物,其中鼠類為其主要貯菌宿主。本菌多樣性的血清型種類以及引起宿主程度不一的症狀,均須經實驗室檢測才能加以確認診斷。目前只要以顯微凝集試驗(microscopic agglutination test, MAT)做為宿主鉤端螺旋體症之抗體檢測標準方法,此方法乃以各種不同血清型之活菌做為抗原,因此如用於大量的流行病學調查較為耗費時間與人力,且操作過程需小心謹慎以防感染。與其相比,酵素結合免疫吸附法有著操作較便利且適合大量樣本操作的優勢。Lig蛋白質為近年發表具有做為檢測抗原及疫苗候選者之潛力,只有具致病性的鉤端螺旋體才能表現Lig。因此本研究利用大腸桿菌表現Lig重組蛋白質作為偵測抗原,應用酵素結合免疫吸附法(enzyme-linked immunosorbent assay, ELISA),評估Lig重組蛋白質在偵測鉤端螺旋體症與現行的顯微凝集試驗結果之一致性。本研究於2008年1月至2009年10月間採集北部與中部兩家屠宰場之屠宰豬隻之血清檢體共1,872份。以顯微凝集試驗利用21株鉤端螺旋體菌檢測,抗體力價大於100倍為陽性標準,總陽性率為51.8% (969/1,872)。力價分布由100倍至51,200倍,主要感染血清群以Australis、 Shermani、Icterohaemorrhagiae為主。以Lig重組蛋白質保守性之區域(LigBCon)做為抗原,以酵素結合免疫吸附法檢測IgG抗體,其敏感性與特異性分別為67.2%以及78.7%,評估其與顯微凝集試驗比較之結果,Kappa值為0.406介於0.4-0.7之間,兩法結果一致性中等。但是當陽性檢體MAT力價低於800倍時,敏感性明顯下低,一致性降低,歸咎於流行病學之抗體盛行率係以IgG為主要抗體,且大部份豬隻皆以低力價感染為主,因此LigBCon-IgG ELISA並不適合取代MAT,用於豬鉤端螺旋體症流行病學調查。
Leptospirosis is an important zoonotic disease worldwide, which is common in tropical and subtropical zones. A variety of serotypes can infect almost all mammas including rodents, they are considered as one of the most important reservoir host. The diagnosis is generally difficult due to diverse clinical manifestation, and often relies on laboratory workup, especially the antibody detection for confirmation. Microscopic agglutination test (MAT) is the gold standard of antibody detection, which requires many serotypes of live bacteria as antigen, it is both time consuming and laborious, not mention there is infection hazard to laboratory personnel. Compare to MAT, enzyme-linked immunosorbent assay (ELISA) is more convenient to operate with bulk samples for epidemiological study. Leptospiral Lig proteins have been identified as a antibody detection antigen and vaccine candidate, and only present in pathogenic species. In this study, we take MAT as standard to evaluate recombinant Lig protein as an immunoglobulin G (IgG) ELISA antigen when used in serological study for swine leptospirosis. A total of 1,872 pig sera were collected from two abattoirs in northern and central Taiwan from January 2008 to October 2009, and examined with a panel of 21 reference leptospira serovars by a MAT. It showed that 51.8% (969/1,872) of sera specimens had a titer of 1:100 or greater to at least one of the serovars employed. The MAT titer range from 1:100 to 1: 51,200. The main reacting serogroups were Australis, Shermani, and Icterohaemorrhagiae. When using recombinant Lig protein conserved region (LigBCon) as ELISA antigen, the sensitivity and specificity were 67.2% and 78.8%, respectively. To evaluate the agreement between MAT and ELISA, the Kappa coefficient were 0.406 (within 0.4-0.7), indicating a fair level of agreement. The sensitivity and Kappa value were further lower when using sera with MAT titer less than 1:800 for calculation. As most of the pigs were infected with low titer; therefore, we conclude that the recombinant Lig protein-based IgG ELISA is not an appropriate MAT alternative for epidemiological survey for swine Leptospirosis.
中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 IX
第一章 前言 1
第二章 文獻回顧 3
一、 鉤端螺旋體簡介 3
(一) 歷史 3
(二) 型態與特性 3
(三) 命名與分類 4
(四) 外膜相關基因(lipL32、lig等) 8
二、 鉤端螺旋體症 10
(一) 傳播途徑 10
(二) 抗體診斷用抗原 13
(三) 臨床症狀 17
(四) 診斷方法 17
(五) 人鉤端螺旋體病流行病學 19
(六) 豬和其他動物鉤端螺旋體症流行病學 22
第三章 材料與方法 26
一、 血清樣本來源 26
二、 顯微凝集試驗(microscopic agglutination test, MAT) 26
三、 LigBCon重組蛋白質抗原之表現與純化 30
四、 LigBCon重組蛋白質抗原之SDS-PAGE電泳分析 31
五、 LigBCon重組蛋白質西方墨點法 32
六、 LigBCon 重組蛋白質之定量 34
七、 酵素結合免疫吸附法(enzyme-linked immunosorbent assay, ELISA)理想條件測試 34
八、 酵素結合免疫吸附法(ELISA)抗體檢測 35
九、 統計分析 36
第四章 結果 37
一、 血清學抗體檢測結果 37
(一) 豬之血清抗體盛行率 37
(二) 陽性血清型分布 37
(三) 血清抗體力價分布 37
二、 LigBCon重組蛋白質表現 41
三、 LigBCon重組蛋白質純化 41
四、 LigBCon-ELISA 44
(一) 理想條件測試 44
(二) cut-off值設定 44
(三) LigBCon-ELISA與MAT之一致性評估 47
第五章 討論 50
參考文獻 54
附錄一 66
LB (Luria – Bertani) medium 66
EMJH (Difco) 66
Enrichment 67
PBS 67
SDS-PAGE膠體配方 68
附錄二 血清檢體檢測結果原始資料 69
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