臺灣博碩士論文加值系統

豬瘟（Classical swine fever；CSF）為一重要且具高度傳染性之疾病。廣泛分佈於世界各地，目前在亞洲大都為地方性疾病且病毒變異性大。快速正確的診斷及治療，可降低豬隻感染豬瘟或其他疾病之死亡率與發病率。豬瘟診斷技術的改善，對於降低感染疾病之風險及疾病爆發之管理是非常重要的。一般豬瘟例行性的診斷包括有酵素結合免疫吸附法（ELISA）、直接螢光抗體染色（DFA）、反轉錄聚合酶鏈鎖反應（RT-PCR）及病毒的分離。應用RT-PCR、原位雜合試驗（ISH）及組織病理學診斷，可用來比較野外感染豬瘟病毒之偵測。本研究以原位雜合反應試驗進行豬瘟病毒之偵測，以非放射性之毛地黃素標記核酸探針，在11個野外感染病例之福馬林包埋組織進行偵測。並抽取扁桃腺檢體之RNA後，以E2基因之廣泛性及特異性引子對其進行RT-PCR試驗。本研究結果顯示，慢性豬瘟感染之病理學研究，以原位雜合反應試驗較RT-PCR及織病理學診斷佳。

Classical Swine Fever (CSF) is a highly infectious and probably the most important disease of swine. Widely distributed around the world, CSF is endemic in much of Asia, where the greatest diversity of viruses is now found. Rapid and accurate diagnosis and prophylaxis are crucial in reducing mortality and morbidity from CSF and other infectious diseases in pigs. Improvements in CSF diagnostics are needed to reduce the risk of disease introduction and to better manage a disease outbreak should it occur. Routine diagnosis of CSF includes the combined use of ELISA, direct immunofluorescence assay (DFA) on tonsil samples, reverse-transcription polymerase chain reaction (RT-PCR), and virus isolation. RT-PCR, ISH and histopathology were compared for the detection of CSFV in naturally infected pigs. In this study, detection of CSFV was studied by in situ hybridization with a nonradioactive digoxigenin-labeled probe in formalin-fixed, paraffin-embedded tissues from 11 naturally infected pigs and RT-PCR from tonsillar samples using both universal and gene specific primers. A set of universal primers and another set of gene specific primers from the E2 gene, which is the major envelope glycoprotein of CSFV, were used in this study for RT-PCR. The ISH technique used in this study proved superior to the other two techniques and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.

CHINESE ABSTRACT…………………………………………………i
ENGLISH ABSTRACT…………………………………………………ii
ACKNOWLEDGEMENTS………………………………………………iii
TABLE OF CONTENTS………………………………………………vi
LIST OF TABLES………………………………………………………ix
LIST OF FIGURES.……………………………………………………x
I. INTRODUCTION………………………………………………………..1
II. LITERATURE REVIEW………………………………………………9
2.1 Background……………………………………………………………9
2.2 The Virus: structure and classification …………………12
2.3 Pathology and Pathogenesis…………………………………….17
2.3.1 Diagnosis……………………………………………………………19
2.3.2 Tentative diagnosis………………………………………………19
2.3.3 Differential diagnosis……………………………………………20
2.3.4 Laboratory diagnosis……………………………………………21
2.3.4.1 Nucleic acid hybridization and PCR techniques…………22
2.3.4.2 Probe preparation………………………………………………27
2.3.4.3 Specimen Preparation………………………………………….29
2.3.4.4 Hybridization……………………………………………………31
2.3.4.5 Detection…………………………………………………………32
2.3.4.6 Controls…………………………………………………………33
III. MATERIALS AND METHODS…………….……………………34
3.1 Animals…………………………………………………………34
3.2 Tissue processing……………………………………………………34
3.3 RNA extraction for probe preparation…………………………35
3.4 Polymerase chain reaction…………………………………………36
3.5 Preparation of labeled probe……………………………………38
3.6 TA-Cloning……………………………………………………………39
3.6.1 Competent E. coli preparation………………………………39
3.6.2 E. coli transformation…………………………………………40
3.7 In situ hybridization……………………………………………44
IV. RESULTS…………………………………………………………47
4.1 Summary of ISH………………………………………………………47
4.2 RNA Extraction………………………………………………………49
4.3 Probe Preparation……………………………………………………52
4.4 In situ hybridization………………………………………………57
4.5 Histopathology………………………………………………………65
V. DISCUSSION…………………………………………………67
VI. CONCLUSION…………………………………………………74
VII. REFERENCES…………………...................……76
VIII. APPENDIX…………………………………………………89
7.1 Appendix 1: In situ Hybridization Buffers………………89 7.2 Appendix 2: In situ Hybridization Protocol……………92 7.3 Appendix 3: Digoxigenin Labelling Protocol……………94
IX. BIOSKETCH OF AUTHOR……………………………………95

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