劉振軒臺灣大學:獸醫學研究所陳幼岭Chen, Yu-LingYu-LingChen2010-05-042018-07-092010-05-042018-07-092008U0001-0207200813570000http://ntur.lib.ntu.edu.tw//handle/246246/178881根據本實驗室歷年來的病理解剖診斷報告，顯示神經系統的病例約佔總病例數的20 %的高比率，且往往與豬隻的高死亡率有關，但因為其幾乎無肉眼可視病變，大多數仍需依靠組織病理學及實驗室檢查以確定其病因。感染中樞神經系統之病原以造成腦炎或腦膜腦炎為主，所呈現的病理變化常為非特異性病變，對病原不易確診。非生物素山葵過氧化酵素 (non-biotin horseradish peroxidase, non-biotin HRP) 免疫組織化學染色是從傳統的免疫化學染色法為基礎發展而來，原理為將染色過程中的卵白素以特殊之化學物質取代，可避免與組織或血液凝集素 (lectin) 形成非特異性的鍵結而造成偽陽性，很適合發展用於病理診斷技術。迴路媒介恆溫增幅法 (loop-mediated isothermal amplification, LAMP), 可將核酸在恆溫下更有效、準確且快速的增幅出來，近年來已成為新興的一種取代聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 的檢測方法。本次實驗共收集84例中樞神經系統疾病感染之豬組織蠟塊，包括豬瘟23例、豬假性狂犬病14例、豬沙門氏菌症5例、豬弓漿蟲症5例、豬第二型鏈球菌感染症24例與未確診疾病13例，其中又將蠟塊年份區分為20年前與近10年兩種，分別利用免疫組織化學染色、聚合酶鏈鎖反應與迴路媒介恆溫增幅法三種方法檢測病原。結果成功建立上述五種豬隻中樞神經系統感染疾病之non-biotin HRP免疫組織化學染色法，且相對於一般傳統染色法，見更強訊號、更容易偵測病原位置的特點，且部分病毒、細菌與原蟲之抗原，均可保存長達20年之久而不被破壞。另外亦成功的自懷疑或確診為感染豬假性狂犬病之27例蠟塊中萃取出組織核酸，並利用PCR方法成功偵測到4例陽性病例，利用LAMP方法成功偵測到8例陽性病例，實驗結果亦成功建立LAMP應用於PRV之診斷技術，也發現較傳統PCR檢測方法來得更敏感且更省時，非常適合應用於快速疾病診斷。Based on the previous studies, histological examination has a limitation to make a definite diagnosis of pathogens of central nervous diseases (CNS). To develop convenient diagnostic tools with high potential for rapid and specific diagnosis for this purpose, non-biotin horseradish peroxidase (non-biotin HRP) immunohistochemistry, loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, and polymerase chain reaction (PCR) are employed in this study. A total of 84 pig cases with miscellaneous CNS diseases were obtained from archival paraffin sections which have been stored up to 20 years. In non-suppurative meningoencephalitis, including 23 pigs with hog cholera, 14 pigs with pseudorabies, 5 pigs with Salmonella sp. infection, 5 pigs with Toxoplasma gondii infection, 13 pigs with unidentified diseases. In suppurative meningoencephalitis, including 24 pigs with Streptococcus suis type 2 meningitis. All cases with any of 5 pathogenic infections were successfully diagnosed by non-biotin HRP immunohistochemistry. DNA from 26 suspected pseudorabies specimens were successful extracted. Among them, 4 samples were amplified and the target nucleic acid was detected by PCR, and 8 samples were amplified and the target nucleic acid was detected by LAMP. The preliminary results demonstrate that non-biotin HRP immunohistochemistry, LAMP, and PCR possess varying potential to detect pathogens of swine CNS diseases for infectious and retrospective studies.目錄--------------------------------------------------------------------------------------- Ⅰ次---------------------------------------------------------------------------------------- Ⅴ次---------------------------------------------------------------------------------------- Ⅶ文摘要---------------------------------------------------------------------------------- Ⅷ文摘要---------------------------------------------------------------------------------- Ⅸ一章 前言-------------------------------------------------------------------------- 1二章 文獻探討--------------------------------------------------------------------- 3一節 豬隻重要神經系統疾病之病原、背景與研究現況---------- 3 2.1.1 豬瘟---------------------------------------------------------- 3 2.1.2 假性狂犬病------------------------------------------------- 4 2.1.3 豬沙氏桿菌症---------------------------------------------- 6 2.1.4 豬第二型鏈球菌------------------------------------------- 6 2.1.5 豬弓漿蟲---------------------------------------------------- 7 2.1.6 重要神經系統疾病診斷現況---------------------------- 9二節 免疫組織化學染色技術------------------------------------------- 9 2.2.1 免疫組織化學染色方法及原理------------------------- 9三節 迴路媒介恆溫增幅法---------------------------------------------- 14 2.3.1 迴路媒介恆溫增幅法之原理---------------------------- 14 2.3.2 迴路媒介恆溫增幅法之產物檢測---------------------- 15 2.3.3 迴路媒介恆溫增幅法之應用與研究現況------------- 15三章 材料與方法---------------------------------------------------------------------- 17一節 實驗設計與流程---------------------------------------------------- 17二節 材料來源------------------------------------------------------------- 18三節 形態病理學檢查---------------------------------------------------- 19　　　3.3.1 組織病理學檢查------------------------------------------- 19 3.3.2 免疫組織化學染色技術---------------------------------- 21四節 分子生物學檢查---------------------------------------------------- 26　3.4.1 陽性假性狂犬病病毒來源 ------------------------------- 26　3.4.2 檢體收集---------------------------------------------------- 26　3.4.3 病毒核酸抽取---------------------------------------------- 26　3.4.4 蠟塊中病毒核酸之製備---------------------------------- 26　3.4.5 16s PCR診斷------------------------------------------------ 26　3.4.6 豬假性狂病PCR診斷------------------------------------- 29　3.4.7 豬假性狂犬病LAMP反應之建立---------------------- 30 3.4.8 PCR與LAMP反應之敏感度比較--------------------- 32四章 結果------------------------------------------------------------------------------- 34一節 形態病理學檢查---------------------------------------------------- 34 4.1.1 豬瘟---------------------------------------------------------- 34 4.1.2 假性狂犬病------------------------------------------------- 35 4.1.3 豬沙氏桿菌症---------------------------------------------- 35 4.1.4 豬弓漿蟲---------------------------------------------------- 36 4.1.5豬第二型鏈球菌-------------------------------------------- 37 4.1.6 未確診之病例蠟塊---------------------------------------- 38二節 分子生物學檢查--------------------------------------------------- 38 4.2.1 16s PCR檢測結果----------------------------------------- 38 4.2.2 PRV PCR檢測結果---------------------------------------- 38 4.2.3 LAMP方法之建立----------------------------------------- 38 4.2.4 PCR與LAMP反應之敏感度比較---------------------- 40五章　討論------------------------------------------------------------------------------- 55一節 　樣本收集分析------------------------------------------------------- 55二節 　組織病理學結果探討---------------------------------------------- 55三節 　傳統免疫組織化學染色法和非生物素過氧化酶免疫組織化學　　　染色法之比較------------------------------------------------------- 56四節 　分子生物學檢查結果--------------------------------------------- 60　5.4.1 16s PCR檢測結果探討----------------------------------- 60　5.4.2 PRV之PCR檢測結果探討------------------------------ 60　5.4.3 LAMP檢測方法之建立----------------------------------- 61　5.4.4 比較PCR與LAMP偵測PRV之敏感度------------- 62　5.4.5 PRV之IHC、PCR與LAMP檢測結果探討--------- 62五節 　結論------------------------------------------------------------------- 63六節 未來工作與展望---------------------------------------------------- 64六章　參考文獻------------------------------------------------------------------------- 65application/pdf3454173 bytesapplication/pdfen-US中樞神經系統疾病非生物素山葵過氧化酵素免疫組織化學染色迴路媒介恆溫增幅法蠟塊Central nervous diseasesNon-biotin horseradish peroxidaseImmunohistochemistryLoop-mediated isothermal amplificationArchival paraffin sections[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178881/1/ntu-97-R95629007-1.pdf