臺灣博碩士論文加值系統

臺灣獼猴 (Macaca cyclopis) 是臺灣唯一的原生特有種非人靈長類動物，其棲地與人類活動範圍高度重疊。由於已有少數血清學研究顯示臺灣獼猴可能有猿猴嗜T淋巴細胞病毒 (simian T-lymphotropic virus, STLV) 感染，且臺灣居民感染人類嗜T淋巴細胞病毒 (human T- lymphotropic virus) 已被證實，因此為探究臺灣獼猴感染人畜共通傳染性之 STLV 的情形，本研究參考Van Dooren等人所發表之文獻，建立半巢式遞減式聚合酶鏈鎖反應 (semi-nested, touchdown polymerase chain reaction) 之STLV前病毒 (provirus) tax 基因序列篩檢技術，並以此技術與市售 ELISA 套組檢測 80 隻臺灣獼猴組織臟器樣本、120 隻臺灣獼猴之EDTA抗凝血液樣本，以及上述 200 隻個體中，128 隻個體的血清樣本。結果顯示 9 隻圈飼與 2 隻野外臺灣獼猴個體中，共 12 件核酸樣本於 PCR 檢測呈 STLV 陽性反應、10 件血清樣本於 ELISA 檢測中呈 HTLV 抗體陽性，個體陽性率為 5.5 % (11/200)。3 個樣本檢出之核酸為首次於獼猴屬個體發現自然感染之第三型 STLV，其餘 9 個核酸樣本則屬於廣泛分布於亞洲與非洲屬獼猴之第一型 STLV。在 7 件陽性組織檢體中，僅生殖系統檢出 2 次，肺臟、骨髓、腸繫膜淋巴結、鼠蹊淋巴結與唾液腺各被檢出 1 次。本研究建立之 STLV 篩檢技術與 ELISA 檢測結果，證實臺灣獼猴之 STLV 感染，並首次於獼猴屬靈長類動物發現自然感染之第三型 STLV。

Formosan macaque (Macaca cyclopis) is the only native species of non-human primate in Taiwan that natural habitat highly overlapped with human activity area. Since previous research revealed the possibility of simian T-lymphotropic virus (STLV) infection in Formosan macaque, and confirmed human infection of human T-lymphotropic virus (HTLV) in Taiwan residents. In order to investigate the infectious status of zoonotic STLV in Formosan macaques in Taiwan, primers of previous studies for tax gene proviral detection had been modified for semi-nested touchdown polymerase chain reaction method establishment. Organ tissues from 80 individuals, EDTA anti-coagulated blood samples from 120 Formosan macaques, and serum samples from 128 individuals among previous 200 individuals had been tested. In 2 wild and 9 captive individuals, 12 samples had STLV tax sequence positive result, and 10 serum samples had HTLV antibody positive result. The overall infectious rate is 5.5% (11/200). The phylogenetic analysis showed 3 neucleic acid samples are STLV-3 which is the first discovery in naturally infected Macaca sp. while the other 9 are STLV-1 that had been commonly found in Asian and Afircan macaques. In 7 positive tissues samples, only reproductive system sample had 2 positive results; lung, bone marrow, mesenteric lymph node, inguinal lymph node and salivary gland had only 1 positive result respectively. In this study, the STLV molecular diagnostic method had been established; the combined molecular and serological results confirmed STLV naturally infections in Formosan macaques.

中文摘要 I
Abstract III
謝誌 V
第1章 前言 1
第2章 文獻回顧 3
1.1 非人靈長類之病毒性人畜共通疾病 3
1.2 臺灣獼猴生物學與潛在人畜共通疾病風險 3
1.2.1 臺灣獼猴生物學簡介 3
1.2.2 潛在人畜共通疾病風險 4
1.3 臺灣獼猴傳染性疾病調查 5
2.1非人靈長類之反轉錄病毒 6
2.2 靈長類嗜T淋巴細胞病毒 (primate T-lymphotropic virus) 7
2.2.1 命名分類學 7
2.2.2 PTLV基因、蛋白質與複製 8
2.2.3 PTLV 親緣關係 9
2.2.4 PTLV實驗室診斷 10
2.2.4.1 病毒分離 10
2.2.4.2 血清學診斷 10
2.2.4.3 分子病毒學診斷 11
2.2.5 PTLV 於野外感染概況 14
2.2.6 PTLV之傳播模式 17
2.2.7 PTLV之人畜共通傳染 19
第3章 材料與方法 20
3.1 實驗設計 20
3.2 研究樣本 20
3.2.1 臺灣獼猴樣本來源 20
3.2.3 樣本類型 21
3.2.4 樣本數量 22
3.2.5 血液樣本採集方式 22
3.2.6 組織臟器樣本採集方式 24
3.3 樣本前處理 24
3.3.1 血液樣本前處理與保存 24
3.3.2 組織臟器樣本前處理與保存 24
3.4 核酸萃取 25
3.5 STLV之半巢式聚合酶鏈鎖反應 ( semi-nested polymerase chain reaction; semi nested PCR ) 的建立 25
3.5.1 引子設計 25
3.5.2 條件控制組 (condition control) 序列合成 26
3.5.3 樣本篩檢之半巢式聚合酶鏈鎖反應 26
3.5.4 樣本篩檢之半巢式遞減式聚合酶鏈鎖反應 (semi-nested, touchdown PCR) 27
3.6 瓊脂膠體電泳分析 (agarose gel electrophoresis) 28
3.6.1 瓊脂膠體製備 28
3.6.2 電泳分析 28
3.7 聚合酶鏈鎖反應產物之純化 29
3.8 定序確認PCR產物 29
3.9 陽性樣本聚合酶鏈鎖反應產物之選殖 29
3.10 陽性樣本質體DNA萃取 31
3.11 陽性樣本質體核酸序列之定序 32
3.12 陽性樣本核酸序列分析 32
3.13 以酵素連結免疫吸附法 (Enzyme-Linked ImmunoSorbent Assay, ELISA) 檢測血清抗體 33
第 4 章 結果 34
4.1　STLV之半巢式聚合酶鏈鎖反應 ( semi-nested polymerase chain reaction; semi nested PCR ) 檢測條件之建立 34
4.1.1　檢測 STLV 之引子對 34
4.1.2 半巢式聚合酶鏈鎖反應之黏合溫度 (annealing temperature) 35
4.1.3 引子對之敏感性測試 35
4.2　陽性對照組之建立 36
4.2.1 血液樣本初步篩檢結果 36
4.2.2 陽性樣本選殖 36
4.2.3 陽性樣本質體之篩檢濃度 37
4.3樣本篩檢之半巢式遞減式聚合酶鏈鎖反應 (semi-nested, touchdown PCR) 37
4.4 臺灣獼猴樣本篩檢 39
4.4.1 篩檢流程與整體篩檢結果 39
4.4.2 圈飼獼猴組織臟器篩檢結果 39
4.4.3 野外獼猴組織臟器篩檢結果 40
4.4.4 圈飼獼猴EDTA抗凝血液樣本之分子生物學檢測 40
4.4.5 野外獼猴EDTA抗凝血液樣本之分子生物學檢測 40
4.4.6 圈飼獼猴血清樣本之ELISA檢測 41
4.4.7 野外獼猴血清樣本之ELISA檢測 41
4.5 STLV核酸陽性樣本與 ELISA 檢測陽性樣本之個體對照 41
4.6 STLV核酸陽性樣本基因序列比對與分析 44
第5章 討 論 46
參考文獻 80
附錄 98
作者簡介 104

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