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研究生:李家豪
研究生(外文):Chia-Hao Lee
論文名稱:建立聚合脢鏈鎖反應與核酸探針技術由福馬林固定組織進行野生動物結核病之診斷
論文名稱(外文):The application of polymerase chain reaction (PCR) and DNA probe on the detection of tuberculosis from fromalin-fixed tissue of zoo animal
指導教授:鄭謙仁;龐飛
指導教授(外文):Chian-Ren Jeng;Victor Fei Pang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:1996
畢業學年度:84
語文別:中文
論文頁數:103
中文關鍵詞:聚合脢鏈鎖反應核酸探針福馬林固定組織野生動物結核病
外文關鍵詞:PCRDNA probeformalin-fixed tissuezoo animaltuberculosis
相關次數:
  • 被引用被引用:2
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結核病的感染目前仍是影響人類與動物健康的重要問題。最近幾年由於後
天免疫不全症候群的流行,非典型分枝桿菌的伺機感染已佔愈來愈重要地
位。同樣的,結核病在牛或野生動物的感染情形也時有所聞。本實驗的目
的為建立以聚合脢鏈鎖反應和核酸探針為主的診斷系統,直接由福馬林固
定組織檢體,進行分枝桿菌的偵測及型別的鑑定。實驗中共使用兩組引動
子,首先以第一組引動子 (PCR-TB) 進行聚合脢鏈鎖反應,可成功地對分
枝桿菌 (包括: M. tuberculosis, M. bovis, M. avium, M.
paratuberculosis 及 M. fortuitum) 65 kD表面抗原基因增幅出一 383
bp 核酸片段,並以非放射線核酸探針配合冷光呈色法確認產物無誤。使
用第二組引動子 (PCR-MT) 也成功地對典型分枝桿菌 (包括: M.
tuberculosis 和M. bovis) 38 kD protein antigen b 基因增幅出一
419 bp核酸片段,並以非放射線核酸探針確認產物無誤。從標準菌株萃取
DNA以十倍連續稀釋進行測試結果顯示,聚合脢鏈鎖反應約可偵測到以1
pg DNA增幅後的產物,核酸探針則可更進一步偵測到以 100 fg DNA增幅
後的產物。在過去由臺北市立動物園進行野生動物病理剖檢所收集的53個
疑似結核病感染福馬林固定組織檢體中,抗酸性染色呈陽性有32例,陽性
率為 60.37%;PCR-TB反應陽性有37例,陽性率為69.81%,Probe-TB呈
陽性反應有41例,陽性率為77.36%;PCR-MT反應陽性有32例,陽性率
為60.37%間AProbe-MT陽性反應有33例,陽性率為62.26%。在抗酸性染
色呈陽性的32個病例中,經聚合脢鏈鎖反應確認為人型/牛型分枝桿菌感
染的有23例,以核酸探針偵測確定為人型/牛型分枝桿菌感染的有25例。
實驗結果顯示目前建立之聚合脢鏈鎖反應與非放射性標定探針診斷系統,
對於無法進行細菌培養鑑定的檢體,將會是結核病診斷上重要的輔助工具

Mycobacterial infection is an important problem of zoonosis. O-
pportunistic infections with atypical mycobacteria in the
course of AIDS gained importance in the past decade. There
have been c- ontinuous occurrences of tuberculosis in bovine
and wild animals. We describe here a assay based on PCR
amplification with 2 gro- ups of primers followed by Southern
blot hybridization with AP- labelled DNA probe to detect and to
group mycobacterial infection in formalin-fixed and tissues of
zoo animal. A 383 bp fragment e- ncoding for the 65 kD Ag of M.
tuberculosis, M. bovis, M. avium, M. paratuberculosis and M.
fortuitum was dectected by PCR with 1- st group of primers. The
specificity of PCR products was confirm- ed by Southern blot
using a region-specific DNA probe. Using the 2nd group of
primers, we achieved specific amplification of a 419 bp
fragment of M. tuberculosis and M. bovis. The product was also
confirmed by Southern blot with DNA probe. Serial dilution
studi- es showed that our PCR method could detect DNA amplified
from 1 pg DNA and the nucleic acid probe we used could detect
DNA ampli- fied from 100 fg DNA. Fifty-three formalin-fixed
tissue blocks of suspecious mycobac- terial infection cases
were collected from Taipei City Zoo. The ratio of positive
reaction for acid-fast staining was 60.4%. Th- e positive
ratio of PCR-TB and Probe-TB were 69.8% and 77.4%; the
positive ratios of PCR-MT and Probe-MT were 60.4% and Within
the 32 cases showing positive result, there were 23 and 25
cases are belonged to the typical mycobacterial infection
detect- ed by PCR and confirmed by Southern blot with AP-
labelled nucleic acid probe. The result indicated that the
methods of PCR and Sou- thern blot hybridization described
above should be a important di agnostic method in detecting and
grouping mycobacterial infection in formalin-fixed tissues.
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