臺灣博碩士論文加值系統

犬瘟熱病毒(canine distemper virus；CDV)屬於Paramyxoviridae科，Morbillivirus屬，感染犬隻後會引起呼吸道、消化道以及神經症狀，尤其對 3-6 月齡幼犬引發高發生率及高死亡率。雖然透過疫苗注射的接種本病普遍可獲得良好的控制，但國內外仍時有病例發生且近年來有增加之趨勢，本病的早期症狀與犬舍咳很難區分，因此造成疾病早期診斷上的困難。為建立快速與敏感之診斷本實驗藉由 RT-PCR 結合巢式聚合酶鏈鎖反應 (Nested PCR) 針對臨床感染疑似之病例進行檢測。在本實驗共收集了 440 個臨床上懷疑 CDV 感染病例共 661 個檢體，檢體種類包含全血、眼結膜、口、鼻拭子以及尿液和直腸拭子。檢測結果發現 Nested PCR 之陽性率為37.7% (166/440) 比 RT-PCR 17.0% (75/489) 增加 20.7% 診斷的有效性 (P<0.001)。統計在感染季節分佈方面，各月份均有 CDV 感染之病例，其中以晚秋及冬季發生率較高(P<0.001)。為建立一個有效的檢體採樣選擇，將 Nested PCR 診斷為陽性的 166 個病例中具有多重檢體的 62 個病例做進一步分析，發現檢出率以眼結膜抹片 100.0% (11/11) 為最高，比血液 64.4% (38/59) 高出甚多(P<0.05)。在Real-time PCR 定量 27個病例各臨床檢體的病毒量，結果發現臨床檢體中犬瘟熱病毒的定量最高可達1.8 x 1010 copies/mL，而單一病例檢體間病毒量的差異可高達 109倍。在與臨床症狀相關性分析方面，於眼結膜拭子、鼻腔拭子、口腔拭子、直腸拭子及尿液等檢體均可定出相當高之病毒量，證明病毒可經由呼吸道、消化道及泌尿道排毒。而不論在病毒血症或非病毒血症的病例，大多數的鼻腔拭子具有較高的病毒量。另外有 1/3 (9/27) 血液檢測為陰性之病例，於其它檢體中仍可測得相當高的病毒量，可見血液並不是最佳的檢測檢體。本實驗結合62 個具有多重檢體的陽性病例分析與病毒定量之結果，犬瘟熱感染犬隻臨床檢體之採樣須考量採樣當時病犬之臨床症狀，才可達到最佳之檢出率。臨床上具全身性症狀之犬隻其眼結膜抹片及眼、鼻分泌液檢體具有較高的檢出率，另外患犬若有消化道症狀其直腸拭子亦有相當高的檢出率，具神經症狀之犬隻則以尿液及眼結膜抹片檢體檢出率較高，若患犬有發熱及流涎之症狀其血液與唾液檢體亦是不錯的考量，而利用RT-nPCR可達到最佳的檢出效果。 H 基因序列分析結果顯示，目前 CDV 台灣株間基因相似度介於 99.1%-99.5% 之間，與鄰近國家日本 1992 年犬 Hammam 分離株的基因相似度則介於 98.9 %-99.0% 之間皆屬於 Asia-1 基因型。

Canine distemper virus (CDV) infection in dogs can result in subclinical infection, gastrointestinal signs, and/or respiratory signs, frequently with central nervous system (CNS) involvement, high morbidity and mortality. Recently, the incidence of canine distemper (CD) both in unvaccinated and vaccinated dogs seemed increasing in Taiwan. In order to understand more about the current CDV infection in Taiwan, a rapid and sensitive diagnotic test for CD using a RT-PCR combined with nested PCR was applied to 440 dogs clinically suspected with CDV infection. 660 clinical specimens including CSF, whole blood, nasal swab, ocular swab, rectal swab and urine were collected from 440 dogs. The results showed that Nested PCR (37.7%, 166/440) increase 20.7% in the efficiency of the diagnosis than RT-PCR (17.0%, 75/489) (P<0.001). The seasonal distribution of the infection was mainly seen in late autumn to winter (P<0.001). Detection rate of conjunctival scraping (100.0%, 11/11) had a significant elevation compared with whole blood (64.4%, 38/59) (P<0.05). Quantification of the viral RNA concentration in clinical specimens from 27 cases by real-time PCR revealed that the highest concentration was as high as 1.8 x 1010 copies/mL. Different specimens from the same animal could vary up to 109 times. Nasal swab, ocular swab, oral swab and rectal swab had higher viral load, indicating that virus was shed in respiratory tract, digestive tract and urinary tract correlated to their clinical syndrome. Nasal swab was found to have the highest viral copies in cases with or without viremia. Therefore, the blood is not the best choice for clinical diagnosis. The result of H gene sequence analysis showed, that Taiwanese CDV is in the lineage of CDV Asia-1.

目錄

目錄…………………………………………..………………………………………Ⅰ
圖次…………………………………………………………………………………..Ⅳ
表次…………………………………………………………………………………..Ⅴ
附錄…………………………………………………………………………………..Ⅵ
中文摘要……………………………………………………………………………..Ⅶ
Abstract………………………………………………………………………………Ⅸ
第一章 文獻探討………………………………………..……..……………………1
第一節 犬瘟熱 (Canine distemper, CD)……………………………………1
1.1.1. 犬瘟熱病毒之歷史背景、宿主與流行病學………………………1
1.1.2. 犬瘟熱病毒之病毒特性………………………………...................2
1.1.3. 犬瘟熱病毒之 H (Haemagglutinin) 基因序列分析比對.…..........4
1.1.4. 犬瘟熱病毒感染犬隻之臨床症狀………………………………...5
1.1.5. 犬瘟熱病毒之致病機轉…………………………………………...7
1.1.6. 犬瘟熱病毒感染犬隻之診斷……………………………………...8
第二節 即時偵測、定量聚合酶鏈鎖反應 (Real-time PCR) 技術……….10
1.2.1 Real-time PCR 技術之歷史背景……………………………..…10
1.2.2 Real-time PCR 技術的偵測原理………………………………..11
1.2.3 Real-time PCR 技術的定量分析……………………………..…13
1.2.4 Real-time PCR 技術的融化曲線分析 (Melting curve analysis).14
1.2.5 Real-time PCR 技術的應用……………………………………..14
第二章 序言………………………………………………………………………15
第三章 材料與方法……………………………………………………………....16
第一節 實驗設計與流程圖……………………………………………..…16
第二節 檢體來源…………………………………………………………..16
第三節 寡核苷酸引子與探針之選擇與設計………………………….….17
3.3.1 反轉錄巢式聚合酶鏈鎖反應 (Reverse transcription- nested polymerase chain reaction, RT-nPCR) 寡核苷酸引子之選擇.......17
3.3.2 H 基因分析寡核苷酸引子之選擇………………………….…..17
3.3.3 Real-time PCR 相關寡核苷酸引子與探針之選擇……………..17
第四節 以 RT-nPCR偵測 CDV 核酸……..………………………….….18
3.4.1 病毒RNA的萃取…………………………………………………18
3.4.2 反轉錄反應 (Reverse transcription, RT)………………………....18
3.4.3 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR).………...…..18
3.4.4 巢式聚合酶鏈鎖反應(Nested polymerase chain reaction, nPCR).19
3.4.5 以電泳分析聚合酶鏈鎖反應……………………………………..19
3.4.6 陽性病例之判讀……………………………………………...…...20
3.4.7 利用 Chi-square test與 Fisher’s exact test分析 RT-nPCR 之檢測結果………………………………………………………………..20
第五節 H 基因的選殖與定序…………………………………………….20
3.5.1 具代表性病毒檢體採集時間點之挑選…………………………..20
3.5.2 病毒RNA 之萃取………………………………………………...20
3.5.3 反轉錄反應……………………………..…………………………21
3.5.4 聚合酶鏈鎖反應………………………………..…………………21
3.5.5 H基因之選殖與定序……………………………………………..21
3.5.5.1 接合反應 (ligation)……………………………………….…21
3.5.5.2 選殖質體之轉型作用 (transformation)………………….….22
3.5.5.3 選殖質體之抽取與確認………………………………….….22
3.5.6 犬瘟熱病毒H基因之序列比對分析……………………….……23
第六節 以 Real-time PCR 定量 CDV 核酸………………………….…23
3.6.1 具代表性病例之挑選………………………………………….….23
3.6.2 Real-time PCR 標準曲線之建立…………………………………23
3.6.3 Real-time PCR 之定量分析………………………………………24
第四章 結果………………………………………………………………………25
第一節 以 RT-nPCR 偵測 CDV 核酸…………………………………..25
4.1.1 臨床檢體之檢測…………………………………………………..25
4.1.2 RT-PCR 與 RT-nPCR 之比較………………………………...…25
4.1.3 疑似 CD 病例在各月份陽性率的統計分析………..…………..25
4.1.4 具有多重檢體的陽性病例分析…………………………………..26
第二節 以 Real-time PCR 定量 CDV 核酸…………………………….27
4.2.1 Real-time PCR 標準曲線之建立…………………………………27
4.2.2 Real-time PCR 之定量分析………………………………………27
第三節 H基因之選殖與序列比對分析……………………..……………28
4.3.1 以 RT-PCR 增幅犬瘟熱病毒 H 基因全長……………………..29
4.3.2 經選殖含犬瘟熱病毒 H 基因全長載體之確認……...…………29
4.3.3 犬瘟熱病毒 H 基因核酸序列分析……...………………………29
第五章 討論………………………………………………………………………31
參考文獻……………………………………………………………………………..38









圖次
圖 1. 犬瘟熱病毒之基因模式圖……………………………………..……….……45
圖 2. CDV 相關引子及探針之相關位置圖與增幅基因片段大小……………..…45
圖 3. 以 CDV 上 P 基因特異性引子增幅 CDV 上的特異性 P 基因片段..…46
圖 4. RT-PCR 與 RT-nPCR 比較的柱狀圖……………………………..………..46
圖 5. CDV 在台灣的季節性分佈利用.………………………………….…………47
圖 6. RT-PCR 與 RT-nPCR 於各臨床檢體中偵測犬瘟熱病毒…………….…...47
圖 7. 經選殖含犬瘟熱病毒 P 基因載體之確認……………………..…………...48
圖 8. Real-time PCR 技術偵測已知分子數之連續稀釋含有 P 基因之質體…....48
圖 9. 由已知分子數之連續稀釋含有 P 基因之質體所建立之標準曲線…….....49
圖 10. Real-time PCR 技術偵測已知分子數之連續稀釋含有 P 基因質體之產物電泳圖………………………………………………………...………………49
圖 11. 以Real-time PCR 技術偵測臨床檢體中未知犬瘟熱病毒量…………..…50
圖 12. 以 RT-PCR 增幅犬瘟熱病毒 H 基因全長……………………………….51
圖 13.經選殖含犬瘟熱病毒 H 基因全長載體之確認……………………………51
圖 14. 犬瘟熱病毒 H 蛋白之胺基酸序列比對…………………………………..52
圖 15. 犬瘟熱病毒 H 基因核酸片段之相似百分比及差異性…………………..56
圖 16. 犬瘟熱病毒 H 基因核酸片段之親緣樹狀圖……………………………..57








表次

表 1. CDV P 基因與 H 基因相關引子及探針之核苷酸序列與相關位置…...…58
表 2. 具有多重檢體的陽性病例分析……………………………………………...58
表 3. 臨床症狀顯示為發熱症狀之 CD 陽性病例……..………………………...59
表 4. 臨床症狀顯示為呼吸道症狀之 CD 陽性病例..…………………………...59
表 5. 臨床症狀顯示為全身性症狀之 CD 陽性病例…………………..………...60
表 6. 臨床症狀顯示為全身性症狀同時具有神經症狀之 CD 陽性病例…….....62
表 7. 臨床症狀顯示為神經症狀之 CD 陽性病例…………………………….....63
表 8. 犬瘟熱感染犬隻之年齡、性別與疫苗接種紀錄及臨床檢體之 Real-time PCR定量分析結果…………………………………………………………..64















附錄
附錄 1. 在H基因的監測方面， 1997年即可清楚的看出CDV在不同地區的各自演化結果，主要可區分為日本、歐洲或美國株、 PDV2與疫苗株……………………………………………………………………..…….65
附錄 2. 在日本地區，犬瘟熱病毒自1998年以後至少有兩個基因型同時在日本地區流行Asia/H1與Asia/H2…………………………..………………….66
附錄 3. 2000年在義大利南方捕捉到的四隻6-12月齡紅狐，捕捉後出現神經症狀死亡，經單株抗體證實感染CDV，其H gene經PCR放大之後和在1995年從狗身上分離出的CDV比較，其H gene基因的相似度只有91%..............................................................................................................67
附錄 4. 浣熊感染 CDV 之腦及肺臟組織病理學變化………………..………….68
附錄 5. 美國芝加哥郊區的動物園，於1998年至2001之間感染浣熊的CDV H gene的序列分析樹狀圖…………………………………………………...69
附錄 6. 犬瘟熱致病機制…………………………………………........……...……70
附錄 7. 早期水解探針螢光探針偵測流程圖……………………………………...71
附錄 8. SYBR Green I 螢光偵測流程圖…………….…………………….……..71
附錄 9. TaqMan probe 作用的流程圖………………………...………………....72
附錄 10. 雜合探針作用的流程圖…………………….………….……...…………72
附錄 11. Molecular beacons 作用流程圖………………………………….….…73
附錄 12. Sunrise primer作用流程圖……………………..………………………73
附錄 13. Scorpion Sunrise primer作用流程圖……………..……………………74
附錄 14. Real-time PCR 之定量原理……………………………………………75
附錄 15. 在 Soma et al. 的報告中分別比較檢測血清中 CDV 抗原及 IgM….76

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